Glucose and fatty acid metabolism in infarcted heart from streptozotocin induced diabetic rats after 2 weeks of tissue remodeling

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1 DOI /s y ORIGINAL INVESTIGATION Open Access Glucose nd ftty cid metolism in infrcted hert from streptozotocin induced dietic rts fter 2 weeks of tissue remodeling Christine Mlfitno 1,2*, Alcione Lescno de Souz Junior 2,3, Mrin Cronro 1, Andress Bolsoni Lopes 2, Diego Figuero 1, Lendro Ezequiel de Souz 1, Kleiton Augusto Sntos Silv 4, Fernnd Consolim Colomo 1,5, Rui Curi 2 nd Mri Cludi Irigoyen 1,5 Astrct Bckground: The effects of streptozotocin (STZ)-induced dietes on hert metolism nd function fter myocrdil infrction (MI) remodelling were investigted in rts. Methods: Fifteen dys fter STZ (5 mg/kg.w. i.v.) injection, MI ws induced y surgicl occlusion of the left coronry rtery. Two weeks fter MI induction, contents of glycogen, ATP, free ftty cids nd tricylglycerols (TG) nd enzyme ctivities of glycolysis nd Kres cycle (hexokinse, glucose-6-phosphte dehydrogense, phosphofructokinse, citrte synthse) nd expression of crnitine plmitoyl-coa trnsferse I ( key enzyme of mitochondril ftty cid oxidtion) were mesured in the left ventricle (LV). Plsm glucose, free ftty cids nd tricylglycerol levels were determined. Ejection frction (EF) nd shortening frction (SF) were lso mesured y echocrdiogrphy. Results: Glycogen nd TG contents were incresed (p <.5) wheres ATP content ws decresed in the LV of the non-infrcted dietic group when compred to the control group (p <.5). When compred to infrcted control rts (MI), the dietic infrcted rts (DI) showed (p <.5): incresed plsm glucose nd TG levels, elevted free ftty cid levels nd incresed ctivity of, citrte synthse nd decresed ATP levels in the LV. Infrct size ws smller in the DI group when compred to MI rts (p <.5), nd this ws ssocited with higher EF nd SF (p <.5). Conclusions: Systolic function ws preserved or recovered more efficiently in the hert from dietic rts two weeks fter MI, possily due to the high provision of glucose nd free ftty cids from oth plsm nd hert glycogen nd tricylglycerol stores. Keywords: Glucose metolism, Ftty cid metolism, Dietic hyperglycemi, Myocrdil infrction, Remodeling Bckground Dietic ptients often present crdiomyopthy nd incresed risk of myocrdil infrction, oth of which re ssocited with high rte of mortlity [1]. However, experimentl studies hve demonstrted tht the hyperglycemi in dietic nimls promote chnges in the hert which reduce the intensity of the injury following permnent coronry rtery ligtion. Despite the *Correspondence: chrismlfi@hotmil.com 1 Hypertension Unit, Hert Institute (InCor), Medicl School of University of São Pulo, Av. Enes de Crvlho Aguir, 44, 543 So Pulo, SP, Brzil Full list of uthor informtion is ville t the end of the rticle relevnce of these findings, this issue deserves further investigtion. The response of uncontrolled hyperglycemi on hert remodelling fter ischemi is yet to e fully understood [2 8]. Most studies on hert infrction imed to investigte the consequences of the dietic condition in reperfusion. Herts from dietic rts hve een shown to e more sensitive to ischemi/reperfusion in some studies [9, 1] nd to exhiit lower susceptiility in others. In these ltter studies, decresed ftty cid (FFA) oxidtion [11] nd greter glycogenolysis during ischemi [12] re ssocited with reduced myocrdil infrction re [13]. The effects of ischemi/reperfusion on crdioprotection 215 Mlfitno et l. This rticle is distriuted under the terms of the Cretive Commons Attriution 4. Interntionl License ( which permits unrestricted use, distriution, nd reproduction in ny medium, provided you give pproprite credit to the originl uthor(s) nd the source, provide link to the Cretive Commons license, nd indicte if chnges were mde. The Cretive Commons Pulic Domin Dediction wiver ( pulicdomin/zero/1./) pplies to the dt mde ville in this rticle, unless otherwise stted.

2 Pge 2 of 1 re dependent on sher stress, which induces endothelil dysfunction during myocrdil reperfusion [14]. So, the consequences of reperfusion depend on durtion of the preceding ischemi, the hyperglycemic stte, nd [15] on the rte of glucose nd ftty cid oxidtion [16, 17]. Studies involving permnent ischemi nd hyperglycemi on crdioprotection hve shown conflicting results. Exposure to high glucose medium or dietes for short periods of time hs een found to protect the hert ginst permnent ischemi. This my e due to the improved ventriculr function, nd reduced infrcted re nd mortlity reported for dietic rts under ischemic hert injury [7, 8, 18]. Our group hs previously demonstrted tht hyperglycemi my promote the survivl of hert cells y incresing ngiogenesis [7] nd/or y decresing pro-oxidnt dmge. These findings were ssocited to improvement in oth redox lnce nd sympthetic control of the hert function [8]. Fifteen dys fter myocrdil infrction, dietic nimls showed reduced infrct size nd hert firosis re long with improved systolic function, incresed expression of progrmmed cell survivl genes, incresed use of glucose s fuel (s suggested y high GLUT-1 expression), nd reduced intensity of inflmmtion [7]. However, the ssocition etween myocrdil infrction nd metolic chnges in the hert induced y dietes needs to e further investigted. The min purpose of the present study ws to exmine the chnges in glucose nd ftty cid metolism, nd crdic function in streptozotocin-induced dietic rts during tissue remodeling fter myocrdil infrction. The following mesurements were crried out in the lood: FFA, tricylglycerol (TG) nd glucose levels. The following metolic prmeters were mesured in the hert: levels of FFA, TG, ATP, nd glycogen nd ctivities of enzymes of glycolysis nd Kres cycle (hexokinse, glucose-6-phosphte dehydrogense, phosphofructokinse, citrte synthse) nd expression of crnitine plmitoyl- CoA trnsferse I ( key enzyme of ftty cid oxidtion). The hypothesis of the present study ws tht the dietic stte provides more fvorle tissue remodeling condition nd preserves hert function fter MI through chnges in ftty cid nd glucose metolism in the hert. The protocol of ischemi induced y permnent coronry ligtion without reperfusion ws chosen in order to: (1) exclude the jeoprdizing effects of reperfusion per se, such s sher stress nd genertion of rective oxygen species (ROS), (2) void the comintion of other complictions of dietes, such s the rteriosclerosis nd reduced hert lood flow nd (3) exmine tissue remodeling fter injury s n ttempt to estlish new therpeutic pproches in the mngement of ptients who suffered n ischemic hert event. Methods Animls Mle Wistr rts (25 28 g) were otined from the Animl Fcility t the Medicl School of the University of São Pulo, São Pulo, Brzil. The rts received stndrd lortory chow nd wter d liitum nd were housed in cges contining four to five nimls t 22 C in 12-h drk-light cycle. All experimentl procedures were conducted ccording to the Guidelines for the Use nd Cre of Animls Reserch issued y the Ntionl Institute of Helth, nd complied with the ARRIVE guidelines for reporting niml reserch [19]. The protocol used ws pproved y the Ethics in Reserch Committee of the Medicl School of the University of São Pulo. All experimentl procedures were conducted ccording to the Ntionl Institutes of Helth Guidelines for the use nd cre of nimls. The rts were rndomly ssigned to four groups (n = 12 per group): normoglycemic control (C), dietic (D), normoglycemic myocrdil infrcted (MI), nd dietic myocrdil infrcted (DI). The dietic stte (D nd DI groups) ws induced y single i.v. injection of streptozotocin (STZ, 5 mg/kg.w; Sigm Chemicl Co., St. Louis, MO, USA) dissolved in citrte uffer ph 4.5. This experimentl protocol for induction of type I dietes hs een widely used [8, 2 23]. Body weight ws weekly evluted. Blood glucose concentrtion ws mesured to confirm dietes-induced hyperglycemi t the eginning of the 3-dy experimentl protocol, 24 h fter STZ injection (Accu-Check Instnt test, Boehringer Mnnheim, Germny). The normoglycemic rts (the C nd MI groups) received single injection of citrte uffer. Myocrdil infrction ws induced in oth DI nd MI groups, ccording to the procedure descried in the following section. All rts were monitored during 3 dys. The schemtic representtion of the experimentl protocol used is depicted in Fig. 1. Fifteen dys fter STZ injection or dministrtion of citrte uffer, MI nd DI rts were nesthetized (8 mg/ kg.w. ketmine nd 12 mg/kg.w. xylzine, i.p.) nd underwent myocrdil infrction y surgicl occlusion of the left coronry rtery s previously descried in severl studies [7, 8, 18, 22, 24]. C nd D nimls underwent shm surgery with no induction of myocrdil infrction in order to determine the effects of the surgicl procedure itself. Echocrdiogrphic mesurements in the hert Echocrdiogrphy ws performed t the end of the experimentl protocol y n oserver linded to group lloction, ccording to the guidelines of the Americn Society of Echocrdiogrphy. Rts were nesthetized s descried ove nd imges were otined with

3 Pge 3 of 1 1 Dy 15 Dy 3 Dy Injectionof: STZ inthe dietic groups (D nd DI); Citrte uffer inthe normoglycemic groups(c nd MI). Induction of myocrdil infrction in the groups: MI nddi. Thertsofll groups(c, D, MI nd DI) were Killed. Fig. 1 Experimentl protocol used to study the effects of myocrdil infrction nd tissue remodelling on crdic function nd hert glucose nd ftty cid metolism in dietic nd control rts 1-14 mhz liner trnsducer in SEQUOIA 512 (ACU- SON Corportion, Mountin View, CA, USA) for mesurement of systolic function (ejection frction-ef nd frctionl shortening-fs), s descried in detil elsewhere [23, 24]. MI size ws estimted on the sis of kinesis or dyskinesis res. Regions with systolic shortening were clssified s norml, hypokinetic or sent (kinetic), nd sections with prdoxicl movement were considered dyskinetic. Myocrdil infrction size ws estimted s the rtio of the infrcted re to the totl perimeter of the endocrdil order of the LV (%) [25]. To confirm the echocrdiogrphic mesurements, MI size ws lso evluted y dissecting the firous scr from the remining LV muscle [7]. Experimentl procedure The nimls were euthnized y decpittion fter 15 dys of the myocrdil infrction surgery or 3 dys of induction of dietes (Fig. 1). Plsm nd left ventricle were removed nd frozen t 7 C. Determintion of free ftty cids nd tricylglycerols Plsm levels of FFA were evluted using NEFA-HR kit (Wko Dignostics, Richmond, VA, USA). Plsm smples (3 µl) were trnsferred to 96-well plte nd regents (2 µl) were dded following the mnufcturer s instructions. Mesurements of sornce were crried out t 45 nm. Plsm TG concentrtions were determined y enzymtic colorimetric ssy following the mnufcturer s protocol (Bioclin, Belo Horizonte, MG, Brzil). For determintion of TG nd FFA levels in the LV, the hert tissue ws homogenized in chloroform nd methnol solution (2:1). The lower phse ws then trnsferred to new tue nd evported in vcuum centrifuge. Lipid pellets were suspended in 1 µl solute ethnol. The levels of FFA nd TG were determined s descried ove. Determintion of glycogen content in the LV Glycogen content of the LV ws determined y using the ssy descried y Keppler nd Decker [26]. This method is sed on the conversion of glycogen into glucose y myloglucosidse (Sigm,St. Louis. MO, USA). Glucose from glycogen ws quntified using glucose determintion kit (Bioclin) nd clculted s mg of glycosyl units/g wet weight of the LV. Assys of the enzyme ctivities LV protein extrcts were sonicted for 3 s, t 4 C, followed y centrifugtion t 1g, t 4 C, for 2 min. Totl protein levels (Brdford method) [27] nd mximl enzyme ctivity were mesured in the superntnts. Hexokinse (HK) ctivity ws determined using the method descried y Crtree nd Newsholme [28]. The extrction uffer contined Tris HCl (5 mm) nd EDTA (1 mm) t ph 7.4. The ssy uffer consisted of Tris HCl (75 mm), MgCl 2 (7.5 mm), EDTA (.8 mm), KCl (1.5 mm), β-mercptoethnol (4 mm), NADP + (.4 mm), ATP (2.5 mm), glucose (1 mm), cretine phosphte (1 mm), cretine kinse (1.8 U/mL), glucose-6-phosphte dehydrogense (1.4 U per ml), nd Triton X-1 (.5 % v/v). The rection ws initited y the ddition of 5 μl glucose (1 mm finl concentrtion) nd sornce mesurement t 34 nm during 1 min. To determine citrte synthse ctivity we used n extrction uffer contining.5 mm Tris HCl nd 1 mm EDTA, ph 7.4, nd n ssy uffer contining 1 mm), DTNB (.2 mm), cetyl-coa (.1 mm), nd Triton X-1 (.1 % v/v), ph 8.1. The rection ws initited y the ddition of 5 µl oxlocetic cid (1 mm finl concentrtion) nd sornce mesurement t 412 nm during 5 min [29].

4 Pge 4 of 1 CPT1 M (crnitine plmitoyltrnsferse I) expression LV protein ws extrcted in uffer contining Trisse (1 mm, ph 7.5); EDTA 1 mm; sodium fluoride 1 mm; sodium pirophosphte 1 mm; sodium ortovndte 1 mm; protinin 1 mg/ml, leupeptin 1 mg/ ml nd PMSF 2 mm) nd quntified y the Brdford method. Electrophoresis ws performed in 7.5 % of sl gel in the presence of sodium dodecyl sulfte (SDS), ccording to the Lemmli method [3]. Electrotrnsference ws performed for 42 min t constnt voltge (4 V) using nitrocellulose memrne (Hyond ECL, GE Helthcre, Sweden). The memrne ws incuted in 5 % non-ft dry milk locking solution for 4 h efore overnight incution t room temperture with polyclonl ntiodies t dilution of 1:1 (Snt Cruz Biotechnology, CA, USA). The susequent steps were performed using the streptvidin/phosphtse lkline system (GE Helthcre, Sweden), nd the nds were reveled using NBT/BCIP sustrtes (Bio Rd Lortories, CA, USA) [31]. Determintion of ATP level in the hert LV ws homogenized in perchloric cid (.6 N) nd the superntnt ws neutrlized using potssium icronte queous solution (1 M). A commercil kit from Invitrogen (Eugene, Oregon, USA) ws used to determine ATP concentrtion in the superntnt. The method is sed on luciferse requirement for ATP in producing light (mximum emission t pproximtely 56 nm nd ph 7.8). ATP levels were clculted s μm nd expressed on the sis of wet tissue weight. A kit sed on luciferin/luciferse ssy hs lso een recently used y other reserchers for determintion of ATP levels [32, 33]. Sttisticl nlysis Results re presented s men ± SEM. One-wy or twowy ANOVA followed y the Student Newmn Keuls post hoc test ws used to compre the groups. Differences were considered significnt t p.5 for ll mesurements. Results Indictors of the estlishment of dietic stte After 3 dys of STZ injection, ody weight ws lowered in dietic rts (D nd DI groups) when compred to normoglycemic nimls (C nd MI groups) (Tle 1). Plsm glucose levels were incresed in STZ-induced dietic rts. MI tissue remodelling did not mrkedly chnge lood glucose levels either in control or dietic rts (Tle 1). Plsm FFA nd tricylglycerol levels were rised in non-infrcted dietic rts (D group) when compred to the other groups (C, MI nd DI). After MI tissue remodelling, plsm FA nd tricylglycerol levels Tle 1 Mesurements of ody weight, glucose, free ftty cids (FFA) nd tricylglycerol (TG) plsmtic levels fter 15 dys of myocrdil infrction nd 3 dys of dietes Groups Body weight Glucose FFA TG C 362 ± ± ± ± 6 D 297 ± 16* 433 ± 25* 21 ± 3* 252 ± 44* MI 378 ± ± 3 15 ± ± 6 DI 35 ± 19* 411 ± 28* 12 ±.9 11 ± 19 Body weight, Glucose, FFA nd TG levels in plsm (mg/dl) of control (C), dietic (D), myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI) rts. Dt re reported s men ± SEM * p.5 compred with C; p.5 compred with MI; p.5 compred with D were decresed in dietic rts (DI group) when compred to non- infrcted dietic rts (D group) (Tle 1). Myocrdil infrct size The myocrdil infrction re of the hert ws significntly smller (24 ± 3 % of the LV perimeter) in the dietic group when compred to the MI group (39 ± 2 % of the LV perimeter) (Fig. 2). Systolic crdic function Systolic function, s indicted y FS (Fig. 2) nd EF (Fig. 2c), ws impired (FS: 25 ± 2 % nd EF: 55 ± 3 %) in the MI group when compred to C (FS: 39 ± 1 % nd EF: 71 ± 1 %) nd D (FS: 39 ± 2 % nd EF: 78 ± 2 %) rts. Systolic function ws preserved fter MI remodelling in dietic rts (FS: 48 ± 2 % nd EF: 82 ± 3 %). Contents of free ftty cids nd tricylglycerols in the left ventricle The free ftty cid levels of the LV were incresed in the DI group when compred to the remining groups (Fig. 3). Tricylglycerol levels in the LV were incresed in the D group when compred to the other groups. After myocrdil infrction, tricylglycerol levels of the LV were mrkedly decresed in dietic rts (DI group) ut remined unltered in the control group (Fig. 3). Glycogen content in the left ventricle Glycogen content of the LV ws incresed in the D group when compred to other rts (Fig. 3c). After MI tissue remodelling, glycogen content in the LV ws decresed in dietic nimls ut remined unltered in the control group (Fig. 3c). Enzyme ctivities of glycolysis nd Kres cycle in the hert The mximum ctivity of G6PDH, phosphofructokinse, HK, nd citrte synthse were mesured. G6PDH nd phosphofructokinse mximum

5 Pge 5 of 1 Myocrdil Infrction Are % MI DI 6 * c 1 * FS % 4 2 * EF% * 2 Fig. 2 Echocrdiogrphic prmeters 15 dys fter myocrdil infrction or fter 3 dys of dietes. Direct myocrdil infrction re mesurement in control myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI) rts. Frctionl shortening nd c Ejection frction in normoglycemic control (C), dietic (D), normoglycemic myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI). Results re presented s men ± SEM of 12 rts for ech group. *p.5 compred with C; p.5 compred with MI; p.5 compred with D ctivity were not mrkedly chnged. HK ctivity ws decresed in the hert of dietic nimls; however, fter infrction nd tissue remodeling (DI group), the vlues reched those of control rts (C nd MI groups) (Fig. 4). Citrte synthse ctivity ws not chnged y dietes. After MI tissue remodeling, citrte synthse ctivity ws reduced in the hert of control rts (MI versus C) ut it remined unchnged in the dietic group (DI versus D). Citrte synthse ctivity, however, ws lower in the hert of DI when compred to C rts (Fig. 4). CPT1 M expression The CPT1-M is n enzyme locted in the inner mitochondril memrne nd plys key role in the control of ftty oxidtion. CPT1-M protein levels were decresed in the hert of D rts when compred to the remining groups. Tissue remodeling olished the decrese in CPT1-M expression oserved in the hert of D rts (DI group), nd vlues reched those of the C nd MI groups (Fig. 5). ATP levels in the hert ATP levels in the hert were decresed in the D, MI, nd DI groups when compred to C rts. This reduction ws more pronounced in DI rts (Fig. 5). Discussion Remodeling plys centrl role in crdic tissue repir nd hs een ssocited to poor MI prognosis [17]. As previously reported y our group, dietic rts show smller hert lesion re fter 15 dys of the MI when compred to normoglycemic nimls. This reduction hs een ssocited with decresed firosis nd incresed expression of cell survivl mrkers, such s vsculr endothelil growth fctor [7], reduction in oxidtive stress nd ttenution of the sympthetic ctivity dysfunction [8]. The present study provides evidence tht tissue remodeling fter MI ws le to preserve/recover more efficiently the systolic function in STZ-induced dietic rts. In this sense, it should e emphsized tht this experimentl model of myocrdil infrction llowed us to nlyze the isolted effect of ischemi on the hert,

6 Pge 6 of 1 mg per g of LV fresh weight * mg per g of LV fresh weight * *. C D MI DI c mg of glycosyl units per g of LV fresh weight C Fig. 3 Free ftty cids; tricylglycerols nd c glycogen contents in the left ventricle of normoglycemic control (C), dietic (D), normoglycemic myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI) rts. Results re presented s men ± SEM of 12 rts per group. *p.5 compred with C; p.5 compred with MI; p.5 compred with D * D MI * DI umol/min per mg of LV fresh weight *.. C D MI DI Fig. 4 Enzyme ctivity in the left ventricle of normoglycemic control (C), dietic (D), normoglycemic myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI) rts: Hexokinse nd. Citrte synthse. Results re presented s men ± SEM of 12 rts per group. *p.5 compred with C; p.5 compred with D umol/min per mg of LV fresh weight * *

7 Pge 7 of CPT1-M (Aritry Units) * # µmol/g of LV fresh weight * * *. Fig. 5 CPT1-M (crnitine plmitoyltrnsferse I) expression nd ATP contents in the left ventricle of normoglycemic control (C), dietic (D), normoglycemic myocrdil infrcted (MI) nd dietic myocrdil infrcted (DI) rts. Results re presented s men ± SEM of 8 rts per group. *p.5 compred with C; p.5 compred with MI; # p.5 compred with DI; p.5 compred with D regrdless of the chronic vsculr complictions usully ssocited with dietic sttes. Also, the jeoprdizing effects of reperfusion, such s sher stress nd ROS production, were prevented. These results reported herein suggest tht hyperglycemi my trigger metolic mechnisms tht would ensure etter remodeling fter ischemic injury. High plsm glucose levels re ssocited with incresed expression of the hypoxi-inducile fctor-1 lph (HIF-1α). This trnscription fctor responds to chnges in oxygen supply nd regultes lood flow nd glucose uptke y incresing crdic tissue vsculriztion prior to ischemi [7]. These chnges seem to ttenute tissue injury under repeted ischemic insults, thus fvoring the consequent remodeling process. As result, new metolic feture is estlished to ensure energy utiliztion efficiency nd norml LV function, nd n ischemic preconditioning-like stte my e estlished in the hert from dietic nimls (Fig. 6). Dietic nimls (D group) showed incresed plsm levels of FFA nd TG nd rised LV levels of TG nd glycogen when compred to the control group efore coronry occlusion (Tle 1; Fig. 3). This metolic feture fvored the remodeling of the ischemic crdic tissue, thus preserving the systolic function. These findings suggest tht in dietic condition the infrcted hert works well fter remodeling under lower ATP level stte (Fig. 5). The mechnisms involved in the hert metolism nd function interreltionship in dietes need further investigtion; however, we might hypothesize tht this ssocition serves to enhnce ATP/ADP-AMP cycling nd to promote chnges in ngiogenesis. We contend tht the chnges in glucose nd ftty cid metolism in the hert induced y dietes re lrgely responsile for the results of systolic function oserved. Tissue remodeling fter MI in the hert of dietic rts somehow enefited from the metolic feture with hyperglycemi, incresed plsm levels of FFA nd TG, ugmented hert glycogen nd tricylglycerol contents, nd reduced hexokinse ctivity, CPT1-M expression, nd ATP levels/in the hert. The metolic profile of the hert in dietic stte involves high uptke nd oxidtion of ftty cids [34]. Rndle et l. [35] hve shown tht incresed plsm FFA concentrtions reduce glucose utiliztion nd cuses glycogen ccumultion in the hert. As previously reported, [36] free ftty cids contriute to decrese glucose oxidtion in the hert fter 6 weeks of streptozotocin-induced dietes. This effect of ftty cids is prtilly reversed y tretment with CPT1 inhiitor. Hirr et l. [37]. hve shown tht mitochondril dysfunction in skeletl muscle cells treted with sturted ftty cids is ssocited with impired glucose metolism nd insulin resistnce, incresed phosphoryltion of Akt nd decresed phosphoryltion of GSK-3, which phosphoryltes nd inhiits glycogen synthse ctivity [38]. Insulin tretment does not chnge either Akt or GSK- 3 phosphoryltion ut increses glycogen content in the hert [39]. The reduced glycogen nd tricylglycerol contents in the LV suggest tht these metolites re redily used when required y the hert. These findings my e ssocited with the chnges in enzyme ctivities of glycolysis, incresed FFA content, nd decresed ATP levels nd CPT1-M expression in the hert. The incresed degrdtion of glycogen nd TG might ccount for the smller infrcted re nd the preservtion of the hert working cpcity nd systolic function found in the dietic group. In fct, stored glycogen nd TG re primry sources of energetic sustrtes to the hert, eing prticulrly importnt in conditions of tissue

8 Pge 8 of 1 1 -M MYOCARDIUM IN DIABETIC STATE - Angiogenesis - REMODELLING fter myocrdil infrction -M c Preference for the use of glucose ssocited to chnges in oxygen supply A new metolic feture is estlished to ensure the energy utiliztion efficiency nd preservtion of the LV function. Dietes is n ischemic precondi oning-like stte Fig. 6 Dietes is n ischemic preconditioning-like stte y incresing the HIF-1α expression, leding to ngiogenesis nd modulting key metolic pthwys to optimize glucose. After myocrdil infrction, this hert is conditioned to increse the expression of glucose trnsporter 1 (Glut- 1), ctivting glycolysis nd FFA uptke (CPT1-M). The O 2 utiliztion in hypoxi to generte sufficient mounts of ATP without producing excessive mounts of ROS c, which would inhiit tricroxylic cid cycle nd mitochondril respirtion, promotes positive remodeling, thus preserving the left ventriculr function. [7]; present dt; c [8] injury nd heling. Rnvigerov et l. [12] hve shown tht fter one-week streptozotocin-induced dietes, rts hve higher glycogen content in the left ventricle nd incresed glycogenolysis ctivity in the hert during ischemi. As result, the hert ecomes more resistnt to ischemi-induced rrhythmis in dietes when compred to controls. Chu et l. [13] hve shown reduced infrcted re nd higher glycogen content in the hert of dietic pigs. Mlfitno et l. [4] nd Egorov et l. [41] hve found tht the increse in glucose vilility, the preferred energetic sustrte for the hert in stress condition, is likely to ply key crdioprotective role under ischemi. Dietes promotes mrked increse in plsm glucose nd free ftty cid levels, thus incresing the vilility of these metolites to tissues nd orgns, including the hert. On the other hnd, the intrcellulr provisions of glucose (from ccumulted glycogen nd plsm) nd FFA (from stored tricylglycerols nd plsm) in the hert from dietic rts led crdiomyocytes to generte oxlocette nd cetyl-coa in order to ensure the intense flux of metolites through the Kres cycle. In ddition to the enhnced provision of glucose nd free ftty cids from the plsm nd inside the cells, increses in citrte synthse ctivity nd in CPT1-M expression were lso oserved in the hert from dietic rts. These results suggest tht the hert then dpts itself to work stisfctorily in low ATP level condition in dietic stte. This mechnism does not work in non-dietic stte. In fct, under this ltter condition, myocrdil infrction remodeling ws ssocited with slight reduction of ATP levels together with impired hert function. Normoglycemic rts without incresed levels of TG nd FFA in plsm nd LV do not develop the postulted ischemic preconditioning-like stte nd utilize energy ineffectively [42]. In the erly stges, crdic remodeling chrcterized y norml or slightly incresed ftty cid (FA) oxidtion plys compenstory, crdioprotective role [43], so new metolic profile is estlished to ensure energy utiliztion efficiency nd the preservtion of LV function (Fig. 6). Peroxisome prolifertor-ctivted receptor (PPAR)-lph is downregulted in ischemic myocrdium nd hs een found to ply role in the switch from ftty cid oxidtion to glucose consumption [44]. In conclusion, the feture of hert glucose together with free ftty cid metolism in dietic nimls efore myocrdil infrction nd remodeling ensure tht the systolic function is preserved/recovered more efficiently when

9 Pge 9 of 1 compred to normoglycemic infrcted nimls. The dietic nimls presented reduced crdic injury nd etter systolic function performnce ssocited with lower ATP levels in the left ventricle. We suggest tht the dietic stte induces chnges in crdic metolism which enhnce the efficiency of tissue remodeling fter ischemic injury. Perspectives Severl therpeutic pproches involving metolism hve een suggested to mitigte the impirment of crdic function cused y myocrdil infrction. For instnce, Hughey et l. [45] hve exmined the efficcy of mesenchyml stem cell trnsplnttion to ttenute the chnges in crdic function nd intermediry metolism found in myocrdil infrction nd insulin resistnce. Evidence is presented herein tht the dietic stte with hyperglycemi nd incresed levels of TG nd FFA in plsm nd hert protects my protect the hert ginst infrction. In fct, hypoglycemi onset s consequence of intensive control of lood glucose levels hs een ssocited with incresed crdiovsculr mortlity [46 49]. Liepinsh et l. [5] hve reported tht the hert is etter protected ginst myocrdil infrction in the postprndil stte due to high insulin-ctivted glucose utiliztion. These oservtions led us to speculte tht the induction of dietic- like metolic condition my represent potentilly novel therpeutic pproch to hndle non dietic ptients undergoing n ischemic hert event. Limittions of study Further studies re needed to elucidte the mechnisms underlying the role plyed y the incresed provision of glucose nd free ftty cids on crdic tissue remodeling fter ischemi. One hs to keep in mind tht the improved crdic function herein reported is proly trnsitory, lsting for short period of time fter MI. The occurrence of neuropthy nd nephropthy in dietic ptients do impir crdiovsculr function if hyperglycemi condition is mintined for prolonged period of time. Authors contriutions CM conceived of the study, cquisition of dt, crried out the iochemistry nlysis, prticipted in the sequence lignment, nd drfted the mnuscript. ALSJ nd MC hve mde sustntil contriutions to cquisition of dt, iochemistry nlysis nd interprettion of dt. ABL prticipted in the nlysis nd interprettion of the dt. DF crried out the echocrdiogrphic nlysis. LES performed the myocrdil infrction surgery. KASS performed the dietes induction. FCC prticipted in the sequence lignment. RC nd MCI conceived of the study, prticipted in the sequence lignment nd drfted the mnuscript. All uthors red nd pproved the finl mnuscript. Author detils 1 Hypertension Unit, Hert Institute (InCor), Medicl School of University of São Pulo, Av. Enes de Crvlho Aguir, 44, 543 So Pulo, SP, Brzil. 2 Deprtment of Physiology nd Biophysics, Institute of Biomedicl Sciences, University of So Pulo, So Pulo, Brzil. 3 Nursing Deprtment, Stte University of Mto Grosso, Alt Florest, Brzil. 4 Deprtment of Nephrology, School of Medicine of the Federl, University of So Pulo, So Pulo, Brzil. 5 Lortory of Trnsltionl Physiology, Universidde Nove de Julho, (UNINOVE), So Pulo, Brzil. Acknowledgements The uthors re grteful to the finncil support of Fundção Zerini, São Pulo, SP; Conselho Ncionl de Desenvolvimento Científico e tecnológico (CNPq: /21-4), São Pulo Reserch Foundtion (FAPESP: 213/ ), nd Guggenheim Foundtion. Competing interests The uthors declre tht they hve no competing interests. Received: 5 August 215 Accepted: 23 Octoer 215 References 1. Roper NA, Bilous RW, Kelly WF, Unwin NC, Connolly VM. Excess mortlity in popultion with dietes nd the impct of mteril deprivtion: longitudinl, popultion sed study. 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