Crdiovsculr Reserch 9 (1998) 81 92 Glucose delivery is mjor determinnt of glucose utilistion in the ischemic myocrdium with residul coronry flow * Lind M. King, Lionel H. Opie MRC/UCT Ischemic Hert Disese Reserch Unit, UCT Medicl School, Observtory, Cpe Town, South Afric Received 24 September 1997; ccepted 2 Februry 1998 Abstrct Bckground: Experimentl dt from isolted rt herts suggest tht glycolysis in severe myocrdil ischemi is inhibited by ccumultion of glycolytic metbolites. In contrst, positron emission tomogrphy (PET) in ptients with myocrdil ischemi records mismtch between the decresed coronry flow in vible ischemic tissue nd n incresed fluorodeoxyglucose ( FDG) signl. To resolve this contrdiction, we investigted glucose uptke t very low coronry flows in the isolted rt hert model. Methods: Rtes of glucose uptke were mesured in the isolted Lngendorff-perfused Wistr rt hert, t control (12 15 ml/ g wet wt/ min) nd low coronry flows (0.1, 0.2 nd 0.5 ml/g wet wt/min) nd t rnge of glucose concentrtions (2.75, 5.5, 11 nd 22 mm). Results: The stedy-stte rte of glucose uptke versus glucose concentrtion could be described by double rectngulr hyperbol t ech coronry flow. Glucose uptke fell to levels significntly below control t low coronry flows. However, the extrction of glucose (glucose uptke s % of glucose delivered) rose shrply, from 1% t control coronry flows, to 25 0% t low coronry flows. Crossover nlysis of glycolytic intermedites in freeze-clmped tissue indicted little inhibition t ny specific site, lthough phosphofructokinse ctivity ws restricted when glycolytic substrte vilbility ws high. Insulin nd preconditioning both incresed glucose uptke with 11 mm glucose, possibly by incresing membrne trnsporter density nd thus incresing glucose delivery to the cytosol. Conclusions: Despite the reduction in bsolute glucose uptke t low coronry flow-rtes, the extrction of glucose ws gretly incresed, possibly following GLUT4 trnsloction. Delivery of glucose to the glycolytic pthwy ppers to be mjor controlling site of glycolysis in low-flow ischemi. Downstrem regultion is then distributed long the pthwy with no one site exerting greter inhibition thn reduced glucose delivery. 1998 Elsevier Science B.V. All rights reserved. Keywords: Glycolysis; Ischemi; Metbolic regultion; Preconditioning; Rt 1. Introduction [6]. The difference between the incresed FDG signl nd the fll in coronry flow is clled metbolism/perfu- Over 20 yers go, Neely nd Rovetto reported tht sion mismtch [7]. These observtions suggest tht bsolute rtes of glucose uptke nd glycolysis re reduced myocrdil glucose uptke could be enhnced in ischemi, in ischemi in isolted herts, phenomenon ttributed to nd tht glycolysis my not be inhibited. Other invesinhibition of the glycolytic pthwy t glycerldehyde-- tigtors hve reched similr conclusions with in vivo phosphte dehydrogense (GAPDH) [1,2] by build-up of models [8], but these concepts hve not been clrified in n metbolites [1, 5]. isolted hert, the model in which glycolytic regultion In contrst, positron emission tomogrphy (PET), which ws initilly described. mesures the trnsfer of F-lbelled deoxyglucose We exmined glucose uptke in the isolted perfused rt ( FDG) into tissue in vivo, shows n incresed FDG hert over rnge of low coronry flows, thought to be uptke in reltion to the fll in the coronry flow-rtes in vible hert muscle of ptients with coronry rtery disese more comprble to in vivo ischemic coronry flow-rtes (defined s those coronry flows found in the subendocrdium of n re perfused by n rtery which is then * Corresponding uthor. Tel.: 144 (65) 275 275; Fx: 144 (65) 275 259; E mil: lind@bioch.ox.c.uk Time for primry review 27 dys. 0008-66/ 98/ $19.00 1998 Elsevier Science B.V. All rights reserved. PII: S0008-66(98)00100-X
82 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 ligted, i.e., 0.07 0.15 ml/g wet wt/min in pigs nd dogs with 11 mm glucose with or without insulin (1 U/l), nd [8]). The crucil issue is whether, when compred to the subjected to 0 min of globl ischemi with no flow, or reduction in coronry flow, the rte of glycolysis is low flow of 0.2 ml/ g wet wt/ min. Two dditionl groups reduced proportiontely, or is upregulted reltive to of herts (n56/ group) (with or without insulin) were coronry flow, s suggested by observtions including preconditioned with 5-min ischemi nd 5-min reperfusion mismtch. We lso investigted the effect of insulin nd prior to low-flow ischemi. Glucose uptke nd lctte preconditioning on glucose uptke nd utilistion within wshout were mesured during low flow ischemi. the cell, both being fctors which my increse trnsloc- Herts were clmped t the onset, fter 15-min nd fter tion of GLUT4 glucose trnsporters to the membrne 0-min ischemi in the glucose nd glucose1insulin [9,10]. groups with Wollenberger tongs kept in liquid nitrogen, for nlysis of tissue metbolites. 6 herts were used for ech time point. Crossover nlysis of the glycolytic inter- 2. Methods medites ws then performed. 2.1. Isolted rt hert perfusions 2.1.. Biochemistry Glucose uptke ws ssessed by the rte of conversion 2.1.1. Experimentl pprtus of glucose 6-phosphte to fructose 6-phosphte, mesured Mle Wistr rts (266 g) fed d libitum (men fresh by the rte of H2O production from D[2- H] glucose [1,]. hert weight 1.0560.01 g) were nesthetised with ether, A trce mount (0.087 mm; 0.2 mci/l) of D[2- H] glucose following which 200 i.u. heprin ws injected into the (Amershm, Amershm, Bucks, UK) ws dded to the exposed femorl vein. The investigtion conforms with the perfuste. The H2O ws seprted from D[2- H] glucose Guide for the Cre nd Use of Lbortory Animls by Dowex columns (Dowex 1X8-200, chloride form, published by the US Ntionl Institutes of Helth (NIH Sigm, St Louis MO), nd the H2O counted in 10-ml publiction No. 85-2, revised 1985). The herts were scintilltion cocktil (Redy-Sfe, Beckmn Instruments, excised, rrested in ice-cold buffer, nd mounted on Fullerton CA). Lngendorff pprtus (perfusion pressure 76 mmhg), Ventriculr tissue ws freeze-dried, nd metbolites perfused with modified Krebs Henseleit solution (in mm extrcted with perchloric cid [1] for determintion of NCl 11.5; KCl 4.75; KH2PO4 1.; NHCO 25, CCl2 high-energy phosphtes nd glycolytic intermedites, or 1.6, N-cette 5) nd gssed with 95% O2 5% CO2 to with ethnol nd NOH for nlysis of glycogen content mintin ph t 7.4. Glucose ws dded to ll perfustes in [14]. The extrcts were then ssyed using spectrophotoconcentrtions s described below. Five millimolr cette metric ssys dpted for use on Cobs Fr centrifugl ws present s n lternte substrte in ll perfusions such nlyser (Roche Dignostics, Berne, Switzerlnd) [1]. tht when glucose ws present only t very low con- Vlues were expressed s mmol/ g wet wt. Glycogen ws centrtions, dverse effects ssocited with limited sub- expressed s mmol 6-crbon units/ g wet wt, fter ssying strte perfusion [11,12] were reduced. In ddition, cette the extrct for glucose. is ftty cid nlogue, the mjor substrte used by the hert. Acette my influence glucose utilistion in normox- 2.2. Expression of results nd curve fitting i. However, our vlues were similr to those obtined in the bsence of cette, especilly in low coronry flow Results were expressed s men6sem. All dt were conditions (dt not shown). expressed s per grm wet weight (wet weight55dry weight from previous determintions). The dt were fitted 2.1.2. Experimentl protocol using Origin (MicroCl, Northmpton, MA) computer A dose-response to glucose ws obtined t rnge of coronry flows. Herts perfused with one of four different glucose concentrtions (2.75, 5.5, 11 nd 22 mm) were subjected to 15 min erobic perfusion, followed by 0 min progrmme.. Results globl ischemi with low coronry flows of 0.1, 0.2 or 0.5 ml/g wet wt/min (n56/group). An infusion pump ws.1. Glucose uptke t different coronry flows nd used to deliver oxygented buffer during low-flow is- glucose concentrtions chemi. The temperture ws mintined t 78C by wter jcketing, nd monitored by thermistor probe in the right.1.1. Chnges in glucose uptke in control nd ischemic ventricle. Coronry efluents were collected for 15 s fter herts 15 min perfusion, nd over ech 5-min period during Preischemic glucose uptke rtes were dependent on the ischemi. Two hundred microlitre smples were tken for perfuste glucose concentrtion, s shown in Figs. 1 nd 2. ssessment of glucose uptke. At ech low coronry flow, glucose uptke fell shrply In second series of experiments, herts were perfused from control levels, nd then incresed to pek t 15 20
L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 8 Fig. 2. Stedy-stte glucose uptke (t 15 min ischemi) versus glucose concentrtion for rnge of coronry flows. At ech coronry flow, glucose uptke ws described by double rectngulr hyperbol, equivlent to Michelis Menten kinetics, with v5v mx[s]/(km1[s]) where v5rte of rection (uptke); [S]5substrte (glucose) concentrtion, Vmx5pprent mximum rte of rection (uptke), Km5[S] where v51/ 2 V (mesure of ffinity where lower K 5incresed ffinity). mx m 2 15 m/g wet wt/min v 5 1.76S/(5.04 1 S), x 5 0.26 2 0.5 m/g wet wt/min v 5 2.S/(11.99 1 S), x 5 6.68 2 0.2 m/g wet wt/min v 5 1.70S/(27.09 1 S), x 5 11.11 2 0.1 m/g wet wt/min v 5 0.60S/(14.61 1 S), x 5 2.6 min ischemi (see Fig. 1). With 2.75 mm glucose, glucose uptke declined fter 15 min ischemi t ech low coronry flow, such tht by 0 min ischemi, the rtes of glucose uptke were similr regrdless of coronry flow (cf. Fig. 1, b, c). With higher glucose concentrtions, glucose uptke ws mintined t plteu level throughout ischemi t ech coronry flow rte (Fig. 1). With coronry flow of 0.5 ml/g wet wt/min, rtes of glucose uptke with 11 nd 22 mm glucose were similr to those in preischemic herts (Fig. 1c)..1.2. Stedy-stte glucose uptke effect of concentrtion nd coronry flow The stedy stte glucose uptke per min, tken s the uptke t 15-min ischemi, ws plotted ginst glucose concentrtion for ech coronry flow. Double rectngulr hyperbolic reltionships were obtined (Fig. 2), equivlent to Michelis Menten kinetics, such tht glucose uptke incresed linerly from 0 5 mm, nd plteued t concentrtions greter thn 11 mm. Similr trends were observed t coronry flows of 0.2 nd 0.5 ml/ g wet Fig. 1. Glucose uptke over time before nd during 0 min low-flow wt/min, s well s with control coronry flows (Fig. 2), ischemi, with different glucose concentrtions t different coronry flows lthough the curves were shifted upwrds, nd to the left () 0.1, (b) 0.2, (c) 0.5 ml/g wet wt/min. Coronry flows in control herts were 15.560.5, 15.60.4, 16.460.6 nd 16.860.6 ml/ g wet wt/ min for with the higher coronry flows. glucose concentrtions of 2.75, 5.5, 11 nd 22 mm respectively (n56 per The right shift in Km for glucose uptke t low coronry group). flows (Fig. 2) suggests tht, in ischemi, higher mini-
84 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 mum glucose concentrtion, of t lest 11 mm glucose, is delivery (mmol/ min/ g wet wt) required to sustin glucose uptke thn is necessry in 5 glucose concentrtion (mmol/ ml) normoxi. The incresed trnsit time of the perfuste through the coronry vsculture with reduced coronry coronry flow (ml/g wet wt/min) (1) flow my led to erly extrction of the vilble glucose, (glucose concentrtion of the perfuste), the term perin the initil portion of the perfused tissue. The con- centge glucose extrction ws derived, centrtion grdient cross the cell membrne t the terminl portion of the vsculture with the lower glucose %Glucose extrction concentrtions would then be insufficient to sustin fcilitted 5 [glucose uptke(mmol/ min/ g wet wt)/ delivery diffusion. This is in greement with stndrd con- (mmol/ min/ g wet wt)] 100 (2) cepts of membrne trnsport kinetics. An dditionl explntion for the difference in glucose When percentge extrction of ech dt point in Fig. uptke observed t low flows nd low glucose concenexponentil reltionship ws found (shown in Fig. b), ws plotted ginst coronry flow, negtive double- trtions is inhomogeneity of flow in the tissue. Some portions of the herts will be subjected to much higher described by: flow thn others, especilly s ischemi progresses. The (2x /1.78) (2x /0.8) %extrction 5 6.81 e 1 14.91 e () steepness of the perfusion grdient will vry depending on 2 the residul flow rte, the glucose concentrtion nd with x5coronry flow (ml/g wet wt/min) nd x 514.91. glucose uptke (determinnts of vibility nd thus of vessel At the norml rnge of coronry flows for n isolted complince), but my result in the sme men glucose perfused hert (12 16 ml/ g wet wt/ min), % extrction uptke s hert with lesser grdient. However, within ws very low, less thn % of tht delivered to the limits, we believe tht we cn drw certin conclusions myocrdium, even though glucose ws the min externl from the dt. This issue is discussed further in Section substrte. There ws shrp increse in glucose uptke t 4.6. coronry flows less thn bout 1 ml/g wet wt/min, from Rtes of glucose uptke nered sturtion t glucose less thn % up to 25 28% t very low flows. concentrtions greter thn 11 mm. Only n increse in When the percentges of glucose extrction for the coronry flow could increse bsolute glucose uptke t different glucose concentrtions (from Fig. 2) were plotted high glucose concentrtions. Thus the mximum rte of ginst delivery of glucose (coronry flowconcentrtion) glucose uptke (pprent V ) ws dependent on coronry rther thn coronry flow, to llow for comprison of the mx flow (Fig. 2). This finding cn be explined by improved dt, similr negtive exponentil reltionship ws found metbolism within the cell nd thus incresed glucose (Fig. c), with shrp increse in glucose extrction t demnd, s well s incresed delivery. reduced glucose delivery. The pttern is the sme for the full rnge of glucose concentrtions..2. Glucose uptke t glucose concentrtion of 11 mm.. The effects of insulin nd preconditioning on glucose uptke nd lctte wshout.2.1. Absolute rtes of glucose uptke The stndrd concentrtion of glucose used in isolted Dt from herts subjected to 0-min low flow ischemi rt hert perfusions in the bsence of insulin is 11 mm. (0.2 ml/g wet wt/min) with 11 mm glucose were com- This concentrtion is twice the physiologicl concentrtion pred to bseline vlues with zero flow ischemi, in the of glucose in plsm nd ensures dequte glucose uptke presence or bsence of insulin (1 U/ l). Insulin incresed in the bsence of insulin nd lternte substrtes. We preischemic glycogen synthesis (21.4061.08 vs plotted the results for 11 mm glucose s function of 16.2760.44 mmol/ g wet wt; p,0.05; Tble 1) s well s coronry flow (Fig. ). ischemic glucose uptke (t 15 min 0.860.1 vs 0.560.1 The vlues t higher coronry flows re open to interpre- mmol/ min; p,0.05; Fig. 4). Lctte production ws ttion, with modultion by oxygen consumption, work rte, incresed in insulin-treted herts (Fig. 4b), lthough lesser insulin nd lternte substrte vilbility. Glucose uptke tissue ccumultion ws found in the low flow herts is not determined by coronry flow under these conditions. becuse of wshout (Tble 1). Totl lctte production At low coronry flows, the dependence of glucose uptke (tissue level1wshout) ws unchnged in low flow vs zero on coronry flow incresed (Fig. ), with fll in glucose flow herts (26.65 mmol vs 27.04 mmol). uptke. Preconditioned herts showed n incresed glucose Absolute glucose uptke is frequently reported in the uptke compred to control herts with only 11 mm literture. However, the uptke of glucose reltive to its glucose (Fig. 4). However, in the presence of insulin, vilbility to the tissue hs not been estblished for preconditioning hd no dditionl effect (Fig. 4). Preisolted rt herts. In order to estblish the reltionship conditioned herts hd lower lctte production (Fig. 4b) between glucose uptke nd delivery of glucose, where: becuse of reduced preischemic glycogen content [15].
L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 85 Fig.. () Glucose uptke versus coronry flow in isolted rt herts perfused with buffer contining 11 mm glucose. The dt is fitted seprtely for low flows (0.1 0.5 ml/g wet wt/min y 5 1.79x 1 0.1; R 5 0.99) nd high flows ( y 520.01x 1 1.28; R 5 0.55). Only t low flows is there significnt reltionship between coronry flow nd glucose uptke. At coronry flows greter thn 12 ml/ g wet wt/ min, fctors other thn coronry flow determine glucose uptke, nd these vlues my vry widely (see Section.2.1). (b) Individul dt points from Fig. () expressed s percentge of glucose delivery i.e. extrction (delivery 5 flow concentrtion), with glucose concentrtion of 11 mm, versus coronry flow. The dt were fitted with (2x /0.8) (2x /14.91) 2 double-negtive exponentil eqution, where y 5 0.87 e 1 6.81 e with x 5 14.91. At flows below 1 ml/g wet wt/min, the percentge extrction increses shrply. (c) Stedy-stte glucose uptke t 15 min ischemi expressed s function of delivery of glucose to llow for different glucose concentrtions nd coronry flows. The percentge extrction for ech point ws then clculted, nd plotted ginst delivery. The dt points from Fig. (b) (11 mm glucose) were lso replotted s function of delivery. The points were then fitted with negtive exponentil reltionship, described by (2x /4.76) (2x /75.42) 2 y 5 20.15 e 1 4.2 e, x 5 14.58.
86 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 Tble 1 Glycogen (mmol C6/ g wet wt), high-energy phosphtes nd glycolytic metbolites (mmol/ g wet wt) in herts perfused with 11 mm glucose with nd without 1 U/l insulin. Herts were clmped t the onset, nd fter 15- nd 0-min zero-flow or low-flow (0.2 ml/g wet wt/min) ischemi (n 5 6/group) Glucose Zero flow Low flow Gluc 1 Insulin Zero flow Low flow Control 15 min 0 min 15 min 0 min Control 15 min 0 min 15 min 0 min Glyc 16.27260.440.85760.700 1.5860.126 5.50561.212 c 6.26660.998 21.9861.08 10.90161.78.85260.662 c 17.06461.620 c 11.8662.260 ATP.48060.21 0.89660.176 0.81660.4 1.74060.0 1.52760.111 2.80960.474 0.8160.464 0.42160.070 1.94960.259 1.62660.281 CP 5.20760.670 0.64260.171 0.81760.244 1.42160.191 1.5160.55 7.0961.861 0.67960.67 0.66560.279 1.25860.108 1.05560.060 G6P 0.08160.015 0.22460.059 0.12560.05 0.09960.044 0.19060.046 0.16760.049 0.46660.077 b 1.11660.21 0.50060.104 0.689160.205 F6P 0.060.00 0.06560.0 0.04460.012 0.0560.011 0.05960.016 0.04060.011 0.1760.02 b 0.760.090 0.14960.05 0.2860.075 FDP 0.07760.01 0.0760.012 0.0560.010 c 0.16260.058 0.05860.014 0.07860.056 0.27660.149 0.04560.0 0.08860.00 0.04660.010 GAP 0.06060.011 0.12860.08 0.10760.05 0.08560.022 0.660.161 0.08260.042 0.17460.050 0.0460.01 0.17160.0 b 0.02560.011 DHAP 0.05860.01 0.02860.005 0.00960.006 c 0.05860.009 b 0.00860.007 0.0560.008 0.15160.075 0.0560.010 0.0260.008 0.01960.008 Pyru 0.07860.005 0.14260.014 0.08860.029 0.101160.009 0.160.01 0.12260.010 0.11560.02 0.1260.010 0.12760.012 0.11760.022 GP 0.28560.082.20660.69 2.58460.748 2.15460.910 2.64460.874 0.87060.1 2.28960.88 4.22860.810.9260.59 4.4460.9 L-Aln.50160.949.19760.45.2560.80 2.98460.16 c 4.97160.90 2.68760.724.11960.54 4.6160.17 c 5.12760.664 4.9460.166 Lct 0.80760.02 24.00660.766 2.75560.740 c 17.15662.252 c 16.27160.56 2.71961.122 2.80262.416 27.04161.105 c 16.54661.0 20.80161.858 %I/(I 1 p) 7.49 2.12 1.6 2.6.12 7.71 4.2 4.55 4.08.68 Glyc: glycogen; ATP: denosine triphosphte, CP: cretine phosphte; G6P: glucose-6-phosphte; F6P: fructose-6-phosphte; FDP: fructose-1,6-diphosphte; GAP: glycerldehyde--phosphte; DHAP: dihydroxycetone phosphte; Pyru: pyruvte; GP: -glycerophosphte (glycerol--phosphte); L-Aln: L-lnine; Lct: lctte. 1,-bisphosphoglycerte, 2-phosphoglycerte nd phosphoenolpyruvte were not mesured s these vlues re very low in tissue [1]. %I/(I 1 P) % intermedite/(intermedite 1 product) where I 5 G6P 1 F6P 1 FDP 1 GAp 1 DHAP 1 Pyru nd P 5GP 1 L-Aln 1 Lct. b c p, 0.05 vs control; p, 0.05 vs 15 min; p, 0.05 vs zero flow..4. Tissue metbolites mintined residul coronry flow, the fll in glycolytic intermedites ws not observed. Glycolysis ws min- Tissue levels of glycolytic intermedites, glycogen nd tined t stedy stte throughout the 0 min ischemic high-energy phosphtes, re shown in Tble 1. Insulin- period, s suggested both by glucose uptke nd metbolite enhnced glucose uptke nd glycogen content, thereby ccumultion. limiting the effect of reduced substrte delivery on gly- The glycolytic intermedites downstrem from glucose colytic flux rtes in ischemi nd llowing regultory sites cn be brodly grouped into intermedites (G6P, F6P, FDP, further downstrem to be indicted. GAP, DHAP, pyruvte) nd end-products (L-lnine, - Glycogen is n importnt contributor to glycolytic flux. glycerophosphte, lctte). The percentge of intermedites The mjority of glycogen is broken down in the initil to intermedites1products declined during ischemi, s minutes of ischemi (12.4 mmol/g wet wt in the first 15 the product ccumultion gretly outweighed tht of the min of glucose zero flow), nd does not relly contribute to intermedites (Tble 1). In ddition, with residul low glycolytic flux fter this time (2.5 mmol/ g wet wt utilised coronry flow, lrge mount of the products, especilly in lst 15 min). The smll mount of residul glycogen lctte, will be wshed out, further reducing the reltive my well be proglycogen, which is more resistnt to intermedite ccumultion. Thus, product formtion gretbrekdown [16], nd is not indictive of glycolytic inhibi- ly outweighs ny intermedite ccumultion, nd negtes tion in ischemi, s hs been suggested [17]. The rte of significnt inhibition t ny point long the pthwy. glycogen brekdown ws ttenuted with low-flow is- GP levels incresed significntly during ischemi, chemi (61% vs. 91% over 0 min), especilly in the indictive of redirection of DHAP to GP, insted of to presence of insulin (44%). GAP nd eventully lctte. However, the mount of GP Anlysis of intermedite ccumultion by the crossover ws significntly less thn tht of lctte. While this theorem [1,] highlighted the ccumultion of G6P, F6P redirection my indicte some inhibition t GAPDH, the nd the end products, lctte nd -glycerophosphte (Fig. 1 GP shuttle is importnt in restoring NAD levels, s is 5). Contrry to previous uthors, who reported increses of the reduction of pyruvte to lctte. An ccumultion of 700% nd 00% in DHAP nd GAP respectively [1], we GP nd restortion of the redox potentil my thus be found no significnt ccumultion of these glycolytic protective mechnism, lthough this is still to be conintermedites. DHAP levels in our herts in fct decresed firmed. significntly during the more severe ischemi used in the In the presence of insulin, G6P nd F6P levels incresed present study (Tble 1). Levels of intermedites tended to gretly during ischemi (Tble 1, Fig. 5), s result of be higher t 15 min thn t 0 min in zero-flow ischemi incresed glycogen nd glucose uptke. Levels of FDP, (Tble 1; Fig. 5), indicting tht glycolysis ws more GAP nd DHAP were lower fter 0 min compred to the limited fter 0 min, with ll vilble intermedites used 15-min vlues in insulin herts, despite high G6P nd F6P up to provide ATP in the bsence of substrte. With vlues, possibly indicting some enzyme inhibition (t
L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 87 Fig. 4. Effect of preconditioning nd insulin on glucose uptke () nd lctte wshout (b) in herts exposed to 0 min low-flow ischemi (0.2 ml/g wet wt/ min). Herts were perfused for 0 min prior to ischemi to llow time for glycogen ccumultion nd the preconditioning protocol. Preconditioned herts were perfused for 20 min, followed by 5-min totl globl ischemi, nd 5-min reperfusion prior to sustined ischemi. Both insulin nd preconditioning incresed glucose uptke, but only insulin hd n effect on lctte wshout. Preconditioning reduces preischemic glycogen, thereby limiting lctte wshout despite incresed glucose uptke. Insulin hd no effect on glucose uptke in preconditioned herts. Lctte is expressed s mmol 6-crbon units (C6)/g wet wt/min, to equte to glucose used. * p, 0.05 ll groups vs glucose control. PFK) with excess glycolytic substrte in the presence of insulin. A bottle-neck effect ws seen, which ws less significnt with residul flow s G6P nd F6P were lower with ttenuted glycogenolysis in the presence of glucose, 1 nd metbolites which could inhibit PFK (i.e. lctte, H ) would be wshed out. Lctte ccumultion ws, however, significntly higher by the end of ischemi in insulin herts, despite ny constriction. The percentge intermedite to totl metbolites ws higher in ll insulin vs. noninsulin herts, lthough there ws still little evidence of significnt GAPDH inhibition. GP tissue ccumultion ws greter in insulin herts, showing more redirection when substrte ws incresed. 4. Discussion We observed tht the extrction of glucose from the perfuste is consistently incresed s the coronry flow is reduced, even to very low vlues. We found n incresed removl of glucose from the perfuste, even though the bsolute glucose uptke ws decresed in severe ischemi.
88 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 glucose detritition. This is gross oversimplifiction, but within the limittions of the technique, we feel tht this ssumption is justified (see Section 4.6). Becuse ccumultion of glycolytic intermedites ws smll compred to rtes of substrte flux through glycolysis, ssessed through end-product ccumultion (Tble 1), we mde the ssumption tht the glucose uptke mesured by D[2 H]-glucose detritition pproximtes glycolytic flux. This pproximtion lso relies on the ssumption tht ll glucose tken up is phosphorylted (see Section 4.6). Thus, for the purposes of the present discussion, glucose entering the cell nd subsequently phosphorylted ws ssumed to be lrgely converted to pyruvte, nd then to lctte under nerobic conditions. Any possible intrcellulr ccumultion of glucose is unlikely to lter the results significntly, given tht we re mesur- ing the rte of the initil steps of glucose utilistion. In ddition, in ischemi ny glucose in the cell is likely to be used up rpidly, s the requirement for ATP increses, nd glycogen synthesis is unlikely. Glycogenolysis contributes substrte to the glycolytic pthwy, the extent of which ws estimted from gross glycogen content in the herts prior to nd t the end of ischemi. Previous clcultions, Fig. 5. Percentge chnge from control vlues of tissue metbolites from herts perfused with 11 mm of glucose with nd without 1 U/l insulin. The bsolute vlues re shown in Tble 1. Herts were clmped t the onset, fter 15 min nd fter 0 min zero or low flow (0.2 ml/g wet wt/ min) ischemi. The dotted line represents control vlues (100%). See Tble 1 for bbrevitions. This incresed removl reltive to the fll in coronry flow supports the observtions of mismtch with FDG in using dt from herts perfused with coronry flow of 0.2 PET studies on dogs [19] nd humns [6,7,20], nd ml/ g wet wt/ min, nd 11 mm glucose [12], show tht observtions of incresed glucose extrction in vivo using glucose uptke nd glycogen utilistion cn ccount for ll different techniques [8]. the lctte produced, which conversely implies tht ll the Our results lso suggest tht the rte of glycolysis in glucose tken up in these conditions is converted to lctte ischemi is determined not so much by the extent of [12]. Residul oxygention cn oxidise only bout 4% enzyme inhibition, but rther by the rte of glucose of glycolytic substrte under these conditions [12]. delivery nd subsequent trnsport into the cell, s previously predicted by computer model [21]. Our results re 4.2. Control vs. regultion of glycolysis in greement with stndrd concepts of substrte product reltionships determining flux through pthwy [22]. We The bove ssumptions invoke the concept of glycolysis do not exclude concomitnt enzyme inhibition, nd hence s metbolon, or single unit mde up of closely some inhibition of ischemic glycolysis occurs s previous- ssocited multiple enzymes [25], which llows efficient ly emphsised, but ny such inhibition ppers of lesser chnnelling of the product of one rection to the next, importnce thn does limittion of glucose delivery by where it becomes the substrte [26]. Thus glycolysis cn very low coronry flows (Fig. 6). Thus, control of gly- be seen conceptully s funnel, the efflux from which, colysis in this model is relted lrgely to substrte supply, is controlled lrgely by the mount of glucose which is lthough there my be downstrem regultion t sites poured into it. The mount of substrte entering the distributed long the glycolytic pthwy [2]. pthwy cn lrgely be ccounted for by the mount tht eventully leves [12] (see Fig. 6). Enzyme inhibition cn 4.1. Substrte supply, glucose uptke nd glycolysis be pictured s constrictions on the funnel spout, leding to some ccumultion of fluid in the funnel, but these re When glucose trnsport is gretly stimulted, phos- not sufficient to prevent the finl outflow. Thus there is phoryltion of glucose by hexokinse, n importnt reg- some regultion of flux by enzyme inhibition distributed ultor of glycolysis when intrcellulr glucose is in excess, long the pthwy, but these individul points do not my become rte-limiting for glycolytic flux [2,24]. pper to be significntly rte-limiting. While PFK my However, we mesured the rte of glucose utilistion hve been inhibited when substrte delivery ws in excess, downstrem of hexokinse. For the purposes of the follow- even when PFK ws mximlly inhibited (insulin nd ing discussion, substrte supply (conceptully mjor low-flow ischemi) the overll rte of glycolysis ws still rte-determining step in enzyme rections [22]) implies very high (estimted by lctte ccumultion). These delivery of substrte vi the coronry vsculture, trnsfer concepts rely on the distinction between control nd into the myocyte, nd the initil phosphoryltion step, s regultion of pthwy, where control is defined s the conversion of G6P to F6P is mesured by D[2- H]- chnge in the level of control fctor exerting chnge in
L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 89 Fig. 6. Possible sites of control nd regultion of glycolysis. (1) Glucose delivery is decresed in ischemi s the coronry flow flls, resulting in reduced bsolute glucose uptke. A reduction in coronry flow below 1 2 ml/ g wet wt/ min ppers to stimulte glucose trnsport, resulting in incresed extrction of glucose from tht delivered to the cells. Glucose uptke nd phosphoryltion re mjor controlling steps of glycolysis, even when these fctors re significntly reduced s in ischemi. (2) Glycogen is rpidly depleted during ischemi (most is utilised within the first 15 min, Tble 1). With totl globl ischemi, or when glucose is present only t very low concentrtions, glycogen is the only significnt glycolytic substrte, nd the tissue content t the onset of ischemi determines subsequent glycolytic rtes. () Phosphofructokinse (PFK) is thought to be the mjor site of glycolytic inhibition under norml conditions when ATP nd CP levels re sufficient. Citrte nd cidic phi re the mjor inhibitors of the enzyme [1,2]. When ATP nd CP levels re depleted, inhibition of PFK is removed llowing glycolysis to continue. Glycolysis is stimulted t this step by fructose 2,6-bisphosphte [41]. However, recent isolted myocyte work nd metbolic control nlysis shows tht in normoxi with sufficient substrte (glucose nd insulin), glucose uptke nd phosphoryltion re the min rte-determining fctors of glycolytic flux [2,24], rther thn PFK. In ischemi, the site of glycolytic regultion ws thought to be GAPDH rther thn PFK [1]. However, if high glucose nd insulin re present, regultion of glycolysis t the level of PFK, possibly due to the drop in phi [1,2] my become of greter importnce. A lrge ccumultion of G6P nd F6P ws found in ischemi, especilly in insulin herts (see Tble 1 Fig. 5), suggesting tht with sufficient or excess substrte, some feedbck is present to prevent too excessive n ccumultion of end product. 1 1 (4) GAPDH ctlyses the conversion of GAP to 2,-bisphosphoglycerte, with the reduction of NAD to NADH 1 H. In ischemi, s lctte ccumultes becuse of incresed production nd reduced wshout, blncing the lctte dehydrogense equilibrium should theoreticlly led to n ccumultion of 1 NADH nd H. Both NADH nd lctte re then thought to inhibit glycolysis t GAPDH [1,2]. However, the importnce of this mechnism in the overll regultion of glycolysis in ischemi is chllenged by the findings tht: (1) glycolysis in ischemi is limited by supply of substrte, in ccordnce with norml substrte product reltionships [22] (Fig. 2); (2) t low coronry flows, glucose extrction is incresed (Fig. b); () hypoxi, ssocited with gretly incresed glycolysis, is lso ssocited with high cytosolic NADH content; (4) regultion of glycolysis in control conditions with excess substrte is limited to less thn 25% t ll steps below phosphoglucoisomerse [2]; nd (5) crossover nlysis of glycolytic intermedites does not point to inhibition t the level of GAPDH (Tble 1 Fig. 5). In ddition, with high glucose t low coronry flows, lctte wshout remins t stedy stte for n extended period, without ny evidence of ttenution of glycolysis by feedbck inhibition [12]. Tissue lctte levels were lso incresed if insulin ws present (Tble 1). While the role of lctte-medited inhibition of glycolysis cnnot be excluded, its importnce ppers reltively smll. Only if excess externl lctte is dded [40], my GAPDH inhibition significntly ttenute the rte of glycolysis, lthough this mechnism hs not been shown directly. The process illustrted in the figure cn be regrded s funnel. Hypotheticlly, in ischemi, if glucose is tken into cell nd phosphorylted, its eventul fte must be lctte if the cell is still vible. Some constriction my be present to slow down the rte, but this does not ffect the eventul outcome. overll flux (e.g. worklod, hormones, substrte concentrtion, coronry flow etc.) while regultion occurs t points within the pthwy which modulte the flux t tht point (e.g enzyme ctivity, cofctors etc) [27]. Our results cll for metbolic control nlysis to determine the sites of regultion of glycolysis in ischemi, s hs been performed
90 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 on glycolysis in normoxic herts perfused with different nd DHAP during ischemi, suggesting tht ny inhibition substrtes [2]. t the level of GAPDH did not hve mjor limiting effect on flux through glycolysis [1, 5] (see Fig. 6). Evidence 4.. Glucose uptke nd extrction versus coronry flow of some inhibition t PFK [1] could be found, especilly in the isolted rt hert when the substrte supply ws gretly incresed by the ddition of insulin (Tble 1). In these conditions, the drop Glucose uptke nd extrction reltive to cute chnges in ph my be greter from incresed glycogen brekdown in coronry flow were investigted using 11 mm glucose [12], such tht cidotic inhibition of PFK is lso greter (with no insulin), s is used in the mjority of isolted rt [2]. However, this inhibition did not significntly limit the hert perfusions. Coronry flow, oxygen consumption nd overll rte of glycolytic flux. The metbolic response of contrctile function re tightly coupled, to regulte hert the tissue to excess substrte dds to the concept of function, nd determine vibility. At norml coronry glycolysis s funnel (Fig. 6); t some point the mount flows in the isolted rt hert (12 16 ml/g wet wt/min), of substrte entering the pthwy my trnsiently exceed glucose uptke is lso modified, by the perfusion model tht which cn exit. However, eventully output will be used, the hert nd work rtes, coronry perfusion pres- incresed becuse of greter ccumulted input. Delivery sure, oxygen vilbility, lternte substrtes nd metbol- of glucose (or vilbility of glycolytic substrte) nd ic sttus of the hert (dibetic, hypertrophied, strved) enzyme inhibition thus compete to determine the finl rte [11,28]. Rtes of glucose uptke re therefore not closely of glucose uptke nd subsequent utilistion. dependent on coronry flow t control coronry flows. However, despite vritions in bsolute glucose uptke 4.4. Does this reltionship occur in vivo? with different models, the percentge extrction of vilble glucose is very low t these flows (Fig. b). At the In ischemi following coronry rtery ligtion, coronry moderte reductions in coronry flows used by other flows in the subendocrdium in lrge nimls re usully uthors (20 60% of norml coronry flows with crys- in the rnge of 0.07 0.15 ml/ g wet wt/ min [8] i.e., bout tlloid perfusions), men glucose uptke vlues my lso 8% of norml coronry flows (1 2 ml/g wet wt/min) vry, but tend to be higher thn those t higher flows [1,4]. [8]. Thus the coronry flows used in the present study This increse cn be ttributed to reversl of the Psteur (0.1 0.5 ml/g wet wt/min, bout 2 8% of norml in vivo effect, with reltive hypoxi, nd fll in citrte nd coronry flows in the rt 5 6 ml/g wet wt/min []) re ATP levels. more physiologicl thn flows of 0.6 ml/g wet wt/min Glucose uptke, nd glycolytic ATP production, is n nd higher s used in previous rt hert studies [1, 5]. In importnt determinnt of vibility, especilly when the the pig hert in vivo, there is no chnge in bsolute glucose hert is compromised by reduced flow [11,12,29]. The uptke s the coronry flow flls to 0.07 0.1 ml/g wet fte of glucose chnges t lower flow (6 ml/ min vs 14 wt/ min but the sme negtive logrithmic reltionship ml/ min [0], s the rtio of nerobic to erobic glucose between glucose extrction nd coronry flow exists [8] s utilistion increses with reduced oxygen vilbility [0]. found in the rt hert. A similr increse in extrction With more severe reductions in coronry flow, s used in reltive to coronry flow occurs in the dog hert with our study, mechnicl function is severely curtiled, or low-flow ischemi induced by LAD ligtion [4]. Thus in bsent, with evidence of ischemic injury [12]. At these the rt, pig [8], dog [10] nd humn hert [7], glucose coronry flows, nerobic glycolysis is the primry source extrction is incresed with impired coronry flow. of ATP [11]. Previously, glycolysis ws thought to be inhibited in severe ischemi becuse glucose utilistion in 4.5. Possible mechnism of incresed glucose extrction n isolted rt hert t coronry flow of 0.6 ml/g wet wt/ min ws less thn tht in the normlly perfused A component of delivery of glucose is the bility of the myocrdium [4]. However, s coronry flow decreses membrne to trnsport glucose, which is determined by the below bout 1 ml/ g wet wt/ min in our model ( is- number of glucose trnsporters in the membrne. Insulin chemi ), the percentge extrction rises shrply (Fig. b), significntly enhnces glucose uptke by incresing the suggesting tht the cpcity of tissue to tke up glucose is density of GLUT4 glucose trnsporters in the membrne, enhnced. Further, glycolytic flux ppers to be limited nd cn thus increse delivery of substrte to the cytosol. lrgely by the vilbility of glucose. A similr prediction Insulin shifts the glucose uptke versus glucose concenws mde with computer modelling t low coronry flows trtion curve significntly upwrds (incresed pprent [21] i.e. tht reduced glucose uptke (nd glycolysis) V mx), indicting tht membrne trnsport becomes rte- results primrily from reduced delivery of substrte [21]. limiting when sufficient substrte is vilble, supporting We hve confirmed these findings with ultr-low coronry the concept tht the initil steps of the glycolytic pth re flows nd rnge of glucose concentrtions in n isolted of mjor importnce in the regultion of glycolysis. The hert. mechnism of incresed glucose extrction in cute is- We did not find evidence of mjor ccumultion of GAP chemi my lso be incresed porosity of the srcolemm
L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 91 medited by trnsloction of glucose trnsporters (GLUT 1 s follows. Only bout 5% of glucose tken up is incorpoor GLUT 4) to the membrne, shown to be triggered by rted into glycogen in the normlly beting hert in the ischemi [9,10]. Insulin cn lso further increse glucose bsence of insulin [9], but this vlue is presumbly even uptke in ischemi, s we hve shown, overcoming the lower in the ischemic hert where ATP formtion is limittion of glucose trnsporter density in the membrne required, rther thn energy storge. An ccumultion of s determinnt of glucose delivery. In the bsence of intermedites my led to disprity between vlues for insulin, only 25 0% of vilble glucose is tken up, glucose uptke nd glycolytic flux (lctte production). leving lrge reserve of glucose. Insulin increses this However, the sum of glycolytic intermedites, other thn extrction to bout 60 70% leving smll reserve, t the products of lctte, GP nd L-lnine, does not which point the grdient of glucose cross the membrne ccount for more thn 7.5% of the totl metbolite limits uptke (Km of GLUT455 mm [5]). Precondition- ccumultion, vlue which decreses during ischemi ing hs lso been shown to induce trnsloction of GLUT4 (see Tble 1, nd from [1]). While GP ccumultes to to the membrne, thereby incresing membrne ffinity for quite lrge extent, the vlues re still smll compred to glucose. We found incresed glucose uptke in precon- both totl glycolytic substrte, nd overll lctte proditioned herts subjected to low-flow ischemi. The lck of duction. Thus these fctors do not significntly ffect the effect of insulin in these herts suggests similr mech- comprison of glucose uptke mesured by D[2- H]-glunism of ction on GLUT4 trnsloction to the membrne. cose with glycolytic flux. However, glucose uptke is not necessrily relted to the 4. An importnt reservtion of our model is tht t very incresed recovery of function in preconditioned herts low coronry flows, the distribution of coronry flow in the [6]. rt hert is likely to be heterogeneous, with consequently Membrne trnsporter ctivity my lso be upregulted differing degrees of cell metbolism nd vibility. Howby fctors ssocited with ischemi including formtion of ever, similr restriction could pply to the studies in the 21 denosine [7], incresed cytosolic C [8] or tissue previous studies of low flow ischemi in the rt hert cyclic AMP [8]. In ddition, contrction-medited glu- [1, 5,11,12,29,0,40] nd investigtions in the humn. cose trnsport differs from insulin-medited trnsport, PET tkes n verge mesurement over volume of indicting the involvement of different pthwys in the tissue, which is similr to glucose extrction by the whole recruitment of glucose trnsporters. Further work is re- isolted rt hert exposed to globl decreses in coronry quired to confirm these hypotheses. Chronic dpttion to flow. However, despite the lck of cellulr homogeneity in ischemi my lso involve chnges in GLUT expression the response of the isolted rt hert to ischemi, the nd distribution. finding of n incresed glucose extrction is supported by lrge niml dt [8,10,19], nd we believe this to be n 4.6. Reservtions importnt observtion. 1. We mesured the mount of D[2- H]-glucose detritited in the phosphoglucoisomerse rection (G6P to 5. Summry F6P), equted to glucose uptke. These dt do not differ significntly from mesurements of rteriovenous glucose The rte of delivery of substrte is mjor determinnt differences (dt not shown), lthough the rdioctive of bsolute glucose uptke t very low coronry flows. methods re more precise. D[2- H]-glucose detritition is While bsolute glucose uptke remins the sme (in vivo), regrded s the gold stndrd for mesuring glucose or flls s the coronry flow flls, the percentge extrction uptke, nd hs lso been used to reflect glycolytic flux increses gretly, indicting n bility of the myocrdium rtes [11,12]. Although use of D[5- H] glucose my be to upregulte its cpcity to trnsport glucose nd thereby more ccurte mesure of glycolysis becuse it is detri- provide ATP essentil for mintined cell function. Similr tited further down the glycolytic pthwy, t enolse, we conclusions hve been reched by others using different found no difference between results from the two isotopes methods of nlysis [8,21,29]. A low coronry flow (,1 in low-flow ischemi (dt not shown). This result lso ml/ g wet wt/ min in the rt hert,10% of norml) thus implies tht the mjority of glucose tken up in ischemi is ppers to trigger n dptive response in the myocrdium, converted to pyruvte. possibly involving trnsloction of glucose trnsporters to 2. D[2- H]-glucose detritition does not distinguish the membrne. between rtes of glucose trnsport cross the srcolemm nd subsequent phosphoryltion. Rtes of phosphoryltion my be ffected by ATP vilbility, which is decresed in Acknowledgements ischemi. This effect needs to be investigted.. Alterntive ftes of glucose, including glycogen nd We wish to cknowledge the contribution of Professor intermedite ccumultion, re not considered in the Dvid Herse, Director of Crdiovsculr Reserch t the overll nlysis of glucose uptke/ glycolysis, for resons Ryne Institute, St Thoms Hospitl, London, in whose
92 L.M. King, L.H. Opie / Crdiovsculr Reserch 9 (1998) 81 92 lbortory the preprtory work ws performed. We would myocrdium determined with fluorine--deoxyglucose. J Nucl Med lso like to thnk Dr Frnçois Boucher for his technicl 1992;:146 15. [20] Tillisch J, Mrshll R, Schelbert H, et l. Reversibility of wll contribution, nd Professor Heinrich Tegtmeyer for his motion bnormlities; preopertive determintion using positron dvice. Lind King cknowledges the finncil support of 1 tomogrphy, fluorodeoxyglucose nd NH (Abstrct). Circultion the University of Cpe Town nd the South Africn 198;68(Suppl III):III 87. Medicl Reserch Council. [21] Kohn MC, Grfinkel D. Computer simultion of entry into glycolysis nd lctte output in the ischemic rt hert. J Mol Cell Crdiol 1978;10:779 796. References [22] Newsholme EA, Crbhee B. Theoreticl principles in the pproches to control of metbolic pthwys nd their ppliction to glycolysis in muscle. J Mol Cell Crdiol 1979;11:89 856. 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Circ trnsition-time nlysis. Trend Biochem Sci 1994;19:19 197. Res 1975;7:7 741. [27] Tegtmeyer H. Energy metbolism of the hert: from bsic concepts [5] Rovetto MJ, Whitmer JT, Neely JR. Comprison of the effects of to clinicl pplictions. Curr Prob Crdiol 1994;19:59 11. noxi nd whole hert ischemi on crbohydrte utilistion in [28] Opie LH, Mnsford KRL, Owen P. Effects of incresed work on isolted working rt herts. Circ Res 197;2:699 711. glycolysis nd denine nucleotides in the perfused hert of norml [6] Mrshll RC, Tillisch JH, Phelps ME, et l. Identifiction nd nd dibetic rts. Biochem J 1971;124:475 490. differentition of resting myocrdil ischemi nd infrction in mn [29] Depre C, Vnoverschelde J-L, Goudemnt J-F, et l. Protection with positron computed tomogrphy, F-lbelled fluorodeoxygluoxide ginst ischemic injury by nonvsoctive concentrtions of nitric cose nd N-1 mmoni. Circultion 198;67:766 778. synthse inhibitors in the perfused rbbit hert. 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