The Control of Sugar Uptake by Metabolic Demand in Isolated Adult Rat Heart Cells

Transcription

1 157 The Cntrl f Sugar Uptake by Metablic Demand in Islated Adult Rat Heart Cells Rbert A. Hawrth and Herbert A. Berkff Frm the Department f Surgery, University f Wiscnsin Clinical Science Center, Madisn, Wiscnsin SUMMARY. T investigate the cntrl f sugar uptake by metablic demand, we used islated quiescent adult rat heart cells in suspensin, under cnditins similar t thse fund during anxia. Metablic demand was varied by expsing cells t rtenne plus varius levels f p- triflurmethxyphenylhydrazne. Withut glucse, the time taken fr half f the cells t underg cntracture was inversely prprtinal t the metablic demand as measured by the rate f lactate prductin. Fr any metablic demand, the nset f cntracture was preceded by a sudden drp in adensine triphsphate. The permeability f cntracted cells t glucse was investigated using 3-0-methylglucse. The rate f 3-0-methylglucse uptake by such cells was strngly dependent n the time taken fr half the cells t underg cntracture, with lw rates at lw times t half cntracture, and insulin-like rates at high times t half cntracture. This suggests that the full inductin f glucse transprt by metablic demand can be prematurely curtailed by the lss f adensine triphsphate. This phenmenn appeared t limit glucse utilizatin in cells with a high metablic demand when glucse was present: such cells underwent cntracture unless insulin was als present, the rate f glucse uptake as measured with 2-dexyglucse was inhibited, and the rate f lactate prductin was inhibited. Isprterenl depressed glucse transprt by tw mechanisms. First, by stimulating the basal metablic demand f the cell it reduced the time taken fr half the cells t underg cntracture and, hence, the level f induced sugar transprt. Secnd, it significantly delayed the nset f sugar permeability with respect t the cntracture event. Cnsequently, cells treated with isprterenl were mre prne t cntracture than cells withut isprterenl. (Circ Res 58: , 1986) Dwnladed frm by n January 11, 2019 EXPOSURE f whle islated rat hearts t perids f anxia results in activatin f glucse transprt (Mrgan et al., 1959). The cmplexity f the whle heart has made it difficult t investigate in detail the way in which glucse uptake is cntrlled under these cnditins. The recent advent f preparatins f islated adult heart cells that resist physilgical levels f calcium has made it pssible t investigate this questin. In this study, anxia was simulated by the additin f rtenne t inhibit mitchndrial respiratin. Varius amunts f p-triflurmethxyphenylhydrazne (FCCP) were added t achieve increased levels f metablic demand. By 'metablic demand,' we mean the rate f adensine triphsphate (ATP) utilizatin by cells sustaining nrmal levels f ATP. Fr such cells, the metablic demand is equal t the rate f ATP prductin. Nrmally, mst ATP resynthesis wuld ccur by xidative phsphrylarin. In the presence f rtenne, ATP prductin is restricted t that frm glyclysis, plus a transient and limited cntributin frm creatine phsphate. Indeed, since there is an bligatry link between glyclysis and ATP prductin, glyclysis cannt prceed unless there is a need fr ATP resynthesis. Thus, if ATP levels are maintained, the steady state rate f lactate prductin becmes a measure f the metablic demand. FCCP is a ptent uncupler f xidative phsphrylatin that is, it inhibits mitchndrial ATP frmatin while (in the absence f rtenne) it stimulates respiratin. Uncuplers als induce mitchndrial adensine triphsphatase (ATPase) activity (Lardy and Wellman, 1953). FCCP in the presence f rtenne increases the rate f ATP utilizatin in islated heart cells by stimulating this ATPase activity, and, since respiratin is inhibited, the rate f lactate prductin is increased (Hawrth et al., 1983). This increase is prprtinal t the amunt f FCCP added, suggesting that, under these cnditins, a unit increase in ATP demand is met by a unit increase in ATP supply (Hawrth et al., 1983). The levels f FCCP used in that study, and in this ne, are extremely lw: the highest level, 6 pml/mg, is apprximately 50-fld less than the level required t uncuple xidative phsphrylatin cmpletely. Since the basal metablic rate f islated cells, being quiescent, is very lw (Hawrth et al., 1983), additin f FCCP is a cnvenient means f increasing the metablic demand in a cntrlled fashin t levels mre typical f cells in a weakly beating anxic heart. We have fund that while glucse uptake is stimulated by cnditins similar t anxia, the extent f stimulatin is curtailed if the metablic demand is t high and this leads t cntracture. Experiments with isprterenl suggest that it can prmte cntracture by this mechanism, by stimu-

2 158 lating metablic demand and cncmitantly reducing glucse transprt. Methds Preparatin f Heart Cells Cell suspensins were prepared as previusly described (Hawrth et al., 1980), except that trypsin was mitted and basal Eagle's medium amin acids (Flw labs) were included at the stage f recirculating perfusin f the hearts, and subsequently until suspensin in the experimental medium. Trypsin was nt used because it was fund t stimulate the basal rate f sugar influx in the absence f insulin, as has als been fund with adipcytes (Pilch et al., 1981). We have previusly shwn that readditin f Ca ++ after heart perfusin is the key t btaining mycytes with a lw in permeability that can resist physilgical Ca ++ levels, and the trypsin additin served t increase the yield f such cells (Hawrth et al., 1982a). The amin acids were used in place f trypsin t increase the yield f Ca ++ -resistant mycytes (Ka et al., 1980). Yields and percent rd-shaped cells were similar t thse previusly described (Hawrth et al., 1980). The rdshaped cells were quiescent in the experimental medium (which cntained 1 mm CaCl 2 ). Incubatin Cnditins Cells were suspended in the experimental medium cntaining: 118 mm NaCl, 4.8 mm KG, 25 mm 4-mrphlinprpane sulfnate (MOPS), 1.2 mm KH 2 PO 4, 1.2 mm MgSO<, 1 mm CaCli, and 2 mm sucrse, adjusted t ph 7.0 with NaOH. They were incubated at 37 C in a Dubnff metablic shaking indicatr set at 90 cycles/min, and allwed t equilibrate with air. The prtein cncentratin was measured at this time by the biuret prcedure and was adjusted t 2.4 mg/ml fr all experiments unless therwise specified. Experiments were initiated typically after 30 minutes f incubatin. Estimatin f Percent Rd-Shaped Cells Three drps f cell suspensin (2.4 mg/ml) were remved at the times shwn in the figures, and mixed with ne drp f 2% glutaraldehyde in the MOPS buffer in rder t fix the cellular cnfiguratin. Fr suspensins f 5 mg/ml, ne drp f suspensin was mixed with tw drps 2% glutaraldehyde. One drp f the mixture was transferred t a glass slide, cvered with a cverslip, and examined with a Zeiss bincular micrscpe at a magnificatin f 320x. The percent rd-shaped cells (i.e., cells with sarcmere lengths in the regin f 1.8 /im) was cunted as described previusly (Hawrth, et al., 1981). Measurement f Sugar Analg Uptake Additins f M C-labeIed sugar analgs and 3 H 2 O were made as described in legends t the figures. We remved 0.5-ml aliquts f cell suspensin at the times shwn, using plastic pipet tips with the ends cut ff s that the pening was abut 2 mm acrss. Aliquts were centrifuged thrugh 0.5 ml f brmddecane int 0.1 ml f 16% perchlric acid, with a Beckman Micrfuge B bench centrifuge. A spin time f 45 secnds was used, althugh the pellet was frmed within secnds. The supernatants were cllected with a Pasteur pipet and pled, and the surface f the brmddecane layer was aspirated free f traces f remaining supernatant. The perchlric acid layer Circulatin Research/Vl. 58, N. 1, January 1986 (& pellet) was remved with a Pasteur pipet, put int 10 ml Aquasl scinn'llatr, and cunted. Supernatant (50 /il) plus an unlabeled cell pellet and 50 fi\ perchlric acid were als cunted fr 10 minutes. After crrectin fr backgrund and verlap f 3 H and U C cunts, results were expressed either as percent permeatin f cell pellet water (fr methylglucse), r uptake in nml/mg (fr dexyglucse). In the case f methylglucse uptake, the supernatant 14 C: 3 H rati defines the 100% value. Thus, when pellet M C: 3 H values are 100%, this means that the [ u C]methylglucse has permeated as much space as the H 2 O, which rapidly equilibrates acrss the cell membrane and determines the ttal aqueus space available. Bichemical Assays ATP was measured in neutralized perchlric acid extracts by the enzymatic assay f Lwry and Passnneau (1972). Lactate was measured in neutralized perchlric acid extracts f aliquts f cell suspensin as previusly described (Hawrth et al., 1983). Presentatin f Data Data shwn usually are frm single representative experiments, since the within-experiment errr was much less than the between-experiment variability. All experiments were dne at least three times. When data are shwn with errr bars r numerically, they are given as the mean ± SD frm at least three experiments. The significance f differences between treatments was evaluated by paired f-test (Wallenstein et al., 1980). Results Cntracture and Metablic Demand First, the effect f glucse, insulin, and isprterenl n the rate f nset f cntracture was investigated. Cntracture was induced by adding rtenne (3 ^M), which cmpletely inhibits mitchndrial NAD-linked respiratin, plus varius levels f FCCP t alter the rate f ATP utilizatin. The change in medium ph was very small (<0.05 ph unit) ver the curse f experiments. With rtenne plus 6 pml FCCP/mg, half f the cells withut glucse underwent cntracture after time ti /2 = 16.5 ± 1.6 minutes withut isprterenl and ti /2 = 13.4 ± 2.0 minutes with isprterenl, and the presence f glucse alne was nt effective in prtecting the cells against cntracture (Fig. 1A). In the presence f insulin, cells withut glucse underwent cntracture with the same time curse as cells withut insulin (Fig. IB). With insulin plus glucse, hwever, the cells were well prtected against cntracture (Fig. IB). When nly 3 pml FCCP/mg were present in additin t rtenne, the nset f cntracture withut insulin r glucse was delayed: ti/2 = 27.9 ± 0.6 minutes withut isprterenl, U/ 2 = 22.5 ± 0.5 minutes with isprterenl. Glucse plus insulin again prtected against cnrracture, but this time glucse alne als gave sme prtectin (data nt shwn). When nly rtenne (n FCCP) was added, the nset f cntracture withut insulin r glucse

3 Hawrth and Berkff/Cntil f Sugar Uptake by Metablic Demand r was als present (data nt shwn). This suggests that, with rtenne alne, the ability f glucse t prtect against cntracture was nt limited by the transprt f glucse acrss the sarclemma, but as the metablic demand is increased by the additin f FCCP, the rate f transfer f glucse acrss the sarclemma increasingly limits the ability f glucse t prtect the cells. Dwnladed frm by n January 11, r FIGURE 1. Effect f glucse, insulin, and isprterenl n cntracture induced by rtenne plus FCCP. Cells in experimental medium were treated with glucse (11 mki ± insulin (1 ^M) 18 minutes befre time zer, ± 1-isprterenl (1 in>i 2 minutes befre time zer, as shwn. Rtenne (3 n>4 was added 2 minutes after time zer, alng with FCCP (6 pml/mg). Cell samples were remved at the times shwn, fixed, and the mrphlgy determined, as described in Methds. Panel A: n insulin; panel B: plus insulin. O, n glucse; A, plus glucse; filled symbls, als plus isprterenl. was delayed further: U /2 = 56.5 ± 0.4 minutes withut isprterenl, ti/ 2 = 39.6± 1.7 minutes with isprterenl. Again, glucse plus insulin prtected against cnttacture, but this time the presence f glucse alne was just as effective as when insulin Cntracture, Lactate Prductin, and Metablic Demand The relatinship between the nset f cntracture and lactate prductin was investigated fr cells withut glucse r insulin. Since n glucse was present, glyclytic ATP came frm glycgen metablism, s 1 ml f lactate is equivalent t 1.5 ml f ATP prduced. Withut rtenne, the rate f lactate prductin was very lw: 0.36 ± 0.05 nml/min per mg withut isprterenl, and 2.00 ± 0.48 nml/ min per mg with isprterenl. With rtenne, the rate f lactate prductin was stimulated, and mre s as mre FCCP was added (Fig. 2, A and B, inset). Hwever, the rate f lactate prductin was always apprximately linear fr a perid, and a similar end pint was bserved at which lactate prductin ceased, regardless f FCCP level (Fig. 2A). The initial linear rate f lactate prductin was linearly related t the level f FCCP added, and the effect f isprterenl was t increase the rate f ATP prductin by a cnstant amunt, independent f the FCCP effect (Fig. 2B, inset). The isprterenl effect, which amunts t 4.70 ± 0.36 nml ATP/min per mg, appears t be a measure f the metablic cst f - stimulatin independent f intrpic effects. When the inverse f the rate f lactate prductin was pltted against the time taken fr half the cells t underg cntracture (t ]/2 frm the experiments in Fig. 1), a linear relatinship was bserved (Fig. 2B), whether isprterenl was present r nt. This suggests that cntracture ccurs after a finite amunt f endgenus substrate has been used up, and that the acceleratin f cntracture by isprterenl is caused by its effect f increasing the rate f cnsumptin f endgenus substrate. ATP and Cntracture When ATP levels were measured in the same suspensins in which cntracture was fllwed, it was fund that ATP levels were maintained at their initial level until a shrt time befre cntracture ccurred, and then declined in parallel with the nset f cntracture (Fig. 3, shwn fr cells with rtenne plus 6 pml FCCP/mg, withut isprterenl). A similar pattern was bserved at all levels f FCCP, with r withut isprterenl, althugh the time lag between half-lss f ATP and half-lss f rd-shaped cells tended t increase with U /2, i.e., as the metablic demand was lwered (Fig. 3, inset). In each instance, the cellular ATP level was maintained cnstant at its riginal level fr a perid,

4 160 Circulatin Research/Vl. 58, N. 1, January O r > I. (cntrcturexmin) ~ 9 5- Dwnladed frm by n January 11, t, Time t Half Cntrcture(min) FIGURE 2. Relatinship f metablic demand, as measured by rate f lactate prductin, t the rate f nset f cntracture withut glucse. Cells in experimental medium (n glucse) were expsed t l-isprterenl (1 /IM), as shwn, 4 minutes befre time zer. Rtenne and FCCP (as shwn) was added at time zer. Samples f cell suspensin (0.2 ml) were remved at the times shwn and added t 0.2 ml f cld 16% perchlric acid. Lactate cntent was then measured as described in Methds. Panel A: lactale accumulatin vs. time; panel B: crrelatin between inverse f rate f lactate prductin and t tn. O; plus rtenne (3 HM), n FCCP; A; plus rtenne, plus FCCP (3 pml/mg); D-Q plus rtenne, plus FCCP (6 pml/mg); diamnds: n rtenne, n FCCP; filled symbls: als plus isprterenl FIGURE 3. Relatinship between cntracture and ATP. Cells were suspended in experimental medium (n glucse) at 5 mg/ml t facilitate ATP measurement. Rtenne (6 ^M) plus FCCP (6 pml/mg) was added at time zer. Samples (0.5 ml) were remved at the times shwn and added t 0.5 ml cld 16% perchlric acid fr ATP analysis, r a few drps were remved and fixed fr mrphlgical analysis. Inset: difference between time f nset f cntracture and ATP lss [t\ n (cntracture) - tip (ATP)], as a funtin f metablic demand [t^n (cntracture)]. N.B. symbls f inset as in figure 2 befre the drp began, and the riginal level was the same at all levels f FCCP, with r withut isprterenl. This justifies ur use f the rate f lactate prductin in the presence f rtenne as a measure f the metablic demand, because the rates are measured at a time when ATP levels are maintained cnstant at their riginal value: since almst all the ATP prduced is cming frm glyclysis, the rate f glyclysis is a measure f the rate at which ATP is being utilized. This suggests that the cells maintain their ATP levels until the endgenus substrate is used up, and then use up mst f their ATP at a rate prprtinal t the metablic demand, befre underging cntracture. We have previusly given evidence that a similar cntracture bserved under cnditins simulating ischemia is caused by a sudden and near ttal lss f cellular ATP (Hawrth et al., 1981). A difference here is that we d nt bserve an initial phase f ATP lss seen under cnditins simulating ischemia (Hawrth et al., 1981). 3-O-Methylglucse Transprt and Metablic Demand T investigate the relatinship f induced glucse transprt t metablic demand, we measured the rate f 3-0-methylglucse uptake by cntracted cells which had undergne cntracture after expsure t rtenne plus varius levels f FCCP, withut glucse. 3-0-methylglucse is a glucse analg which is carried int the cell by the glucse transprter, but which is nt phsphrylated. Transprt is a

5 Hawrth and Berkff/Cntrl f Sugar Uptake by Metablic Demand passive prcess. Cnsequently, 3-0-methylglucse equilibrates with the cell water at a rate gverned by the glucse permeability f the cell membrane (Whitesell and Gliemann, 1979; Hawrth et al., 1984). Lw cncentratins f 3-O-methylglucse («Km) were used in rder t gain a measure f uptake rate uncmplicated by variable rates f back exchange (Whitesell and Gliemann, 1979). At equilibrium, the cncentratin f 3-O-methylglucse inside the cell is equal t that utside the cell. In the uptake assay (Fig. 4A), this is measured as 100% permeatin f the cell pellet, when the rati f pellet "C cunts frm 3-O-methylglucse t pellet 3 H cunts frm 3 H 2 O is equal t the rati f cunts in the supernatant. (Uptake beynd 100% culd suggest a small degree f 3-O-methylglucse binding inside the cell.) The cell pellet cntains sme extracellular medium, and this is allwed fr by measuring the [ 14 C]sucrse-permeable space (Fig. 4A), since sucrse is nt carried int the cell by the glucse transprter. 3-O-methylglucse permeatin beynd the sucrse permeable space thus crrespnds t intracellular penetratin. Figure 4A shws the rate f 3-O-methylglucse uptake by cells cntracted after the additin f rtenne plus 6 pml FCCP/mg. Such cells had undergne cntracture cmpletely after 30 minutes. The rate f 3-O-methylglucse uptake after 35 minutes was slwer in cells expsed t isprterenl than in cells that had nt been expsed (Fig. 4A). If the rate f sugar uptake was measured 15 minutes later, uptake rates were nt significantly changed (Fig. 4A). This suggests that the pst-cntracture rate f sugar entry is fixed. Sucrse was nt fund t enter cntracted cells (Fig. 4A), cnsistent with the cells' cntinued ability t exclude trypan blue (Hawrth et al., 1981). When the rate f 3-O-methylglucse uptake was measured after cntracture induced by rtenne plus different levels f FCCP, it was fund that the rate f entry was strngly dependent n what the metablic demand had been (Fig. 4B). Cells treated with rtenne alne were able t induce a rate f 3- O-methylglucse uptake cmparable t that induced in nrmal cells by insulin. With higher metablic demands, hwever, the rate f induced 3-O-methylglucse uptake was increasingly curtailed. At each metablic demand, the rate f induced 3-O-methylglucse uptake was fund t be independent f the time after cntracture (data nt shwn), like that shwn in Figure 4A. The effect f increasing levels f FCCP cannt be explained as direct inhibitin f 3-O-methylglucse transprt by FCCP: this level f FCCP had n bservable effect n the rate f sugar uptake, either in nrmal cells r in cntracted cells t which FCCP was added after cntracture (data nt shwn). The slwer rates f 3-O-methylglucse uptake bserved with isprterenl (Fig. 4A) culd be explained by the increased metablic rate f such cells, inasmuch as cells with isprterenl shwed a dependence f sugar uptake rate n ti/ 2 similar t that f cells withut isprterenl (Fig. 4B). t 60- a> 1.2 : 0.8 1^8 0.6 IJ r O Time after Rtenne + FCCP Additin (mm) , Time t Half Cntracture (min) 161 FIGURE 4. The dependence f the rate f 3-O-methylglucse uptake by cntracted cells n their previus rate f metablism. A: cells in experimental medium (n glucse) were expsed (9) r nt (O) t isprterenl (1 /IM), 4 minutes befre time zer. At time zer, rtenne (3 nirf plus FCCP (6 pml/mg) was added, alng with 3 H 2 O 0 uci/ ml). 3-O-methylglucse cntaining p 4 Q3-0-methylglucse was added after 35 minutes r 50 minutes, as shwn (180 iim, 0.1 uci/ ml). In ne sample, [ u C]sucrse (0.1 iici/ml) was added at 35 minutes, instead f 3-O-methylglucse. Samples (0.5 ml) were remved at the times shwn and analyzed fr sugar uptake as described in Methds. Panel B: 3-O-methylglucse uptake data frm experiments like that in panel A were fitted t an expnential curve y = a + b Q-e' 1 "), using the intercept fr sucrse uptake as the pint f rigin (aj, and the uptake after 27 minutes as the end-pint (a + bj. The uptake rate 00 was determined fr cells which had undergne cntracture after treatment with rtenne plus varius levels f FCCP. The rates were measured 70 minutes after treatment with rtenne alne, and 40 minutes after treatment with rtenne plus FCCP (3 pml/mg). t m values were taken frm separate experiments (c.f. Fig. 1). Symbls as in Figure 2, except that diamnds represent cells treated with rtenne (3 ^ plus FCCP (100 pml/mg). Dashed line: rate f uptake by nrmal cells plus insulin. Glucse Uptake and Metablic Demand The abve experiments suggest that the inability f glucse t prtect cells against cntracture as metablic demand increases (Fig. 1) is caused by the decreased ability f such cells t transprt the glu-

6 162 cse int the cell where it is needed. Hwever, the experiments shwing a dependence f induced uptake rate n metablic demand (Fig. 4B) were necessarily dne in the absence f glucse. It is pssible that glucse wuld itself amelirate this effect, since any glucse which enters will prmte ATP prductin and, pssibly, ptentiate the further inductin f glucse uptake capability. Unfrtunately, 3-0- methylglucse cannt easily be used in the presence f glucse t measure glucse transprt activity, because the unequal cncentratins f glucse at either side f the membrane causes distrtin f 3- O-methylglucse kinetics. We therefre investigated the effect f metablic demand n glucse uptake by cells in the presence f glucse by using trace levels f 2-dexyglucse (Sklff et al., 1977). 2- Dexyglucse is anther glucse analg that is carried by the glucse transprter. Unlike 3-0-methylglucse, it is phsphrylated inside the cell, but unlike glucse, it is metablized n further (Wick et al., 1957). Since the rate f dephsphrylarin is slw, it tends t accumulate at a linear rate inside the cell (Hawrth et al., 1982b). The disadvantage f 2-dexyglucse is that it des nt simply measure the transprt step. The rate f 2-dexyglucse accumulatin des, hwever, faithfully reprt the rate f glucse phsphrylatin in rat heart (Takala and Hassinen, 1981), and s the rdinate in Figure 5A is labeled 'glucse' uptake. Figure 5A shws that the basal uptake rate fr glucse was very lw (0.21 ± 0.02 nml/min per mg), and was stimulated by rtenne plus varius levels f FCCP, after a lag perid cmparable t the lag perid befre the nset f cntracture withut glucse (Fig. 1). Hwever, the rate f glucse uptake was first stimulated and then inhibited as the metablic demand was increased (Fig. 5A, inset). This uptake rate culd reflect the rate f glucse phsphrylatin by hexkinase (England and Randle, 1967), as well as the rate f glucse entry, but hexkinase will tend t becme mre active, nt less, as the metablic demand increases. The inhibitin f "glucse' uptake seen at high metablic demands (Fig. 5A, inset) is thus cnsistent with the effect f high metablic demands n the rate f 3-O-methylglucse uptake in the absence f glucse (Fig. 4B). The increasing glucse uptake rate with demand seen at lwer demands (Fig. 5A, inset) presumably reflects activatin f the phsphrylatin step, similar t that seen in insulintreated hearts (Mrgan et al., 1961). The effect f isprterenl n glucse uptake at first appears equivcal, giving stimulatin withut FCCP and inhibitin with FCCP. This is nt incnsistent, hwever, since it can be understd in terms f the stimulatin f metablic rate by isprterenl: this stimulatin is apprximately equivalent t the additin f a further 2 pml FCCP/mg (dashed lines, Fig. 5A, inset; c.v. Fig. 2B, inset). We then sught evidence that the inhibited rate f glucse uptake under these cnditins limited the ^ 250 E 20O => 150 O U Circulatin Research/V/. 58, N. 1, January looor FIGURE 5. The effect f metablic demand n glucse uptake and utilizatin. Panel A: cells in experimental medium plus glucse 0 3 mm> were treated with isprterenl (1 MM) as shwn, 4 minutes befre time zer. At time zer rtenne (3 MM) plus FCCP (as shwn) was added, alng with trace levels f ph]2-dexyglucse (1 fici/ml). At the times shwn, 0.5-ml samples were centrifuged (see Methds). Symbls as in Figure 2. Inset: the maximum rate f "glucse' uptake fr each cnditin is pltted. Panel B: effect f glucse and insulin n the rate flactate prductin. Cells in experimental medium with (A, > r withut (O) glucse (11 m\d with (A) r withut <, O) insulin a MM) were expsed t rtenne (3 MM* plus FCCP (6 pml/mg) at t = 10 minutes. Samples were remved at the times shwn and analyzed fr lactate cntent as described in Methds.

7 Hawrth and Berkff/Cntrl f Sugar Uptake by Metablic Demand 163 glyclytic rate. When the lactate prductin f cells treated with rtenne plus 6 pml FCCP/mg in the presence f glucse was measured, it was fund that the rate f lactate prductin became inhibited after 10 minutes, like that f cells withut glucse, althugh the inhibitin was nt quite s cmplete (Fig. 5B). If insulin was als present, the rate f lactate prductin was nt inhibited (Fig. 5B). This shws that the inhibitin f sugar transprt under these cnditins (Figs. 4B and 5A) limited the rate f glyclysis. Chrnlgy f Cntracture and Inductin f Glucse Transprt Since the abve data suggested a pssible crrelatin between the time at which cells withut glucse wuld underg cntracture and the time at which glucse transprt capability is induced, we investigated this relatinship mre clsely by measuring 3-O-methylglucse uptake and the nset f cntracture in the same suspensin f cells. Cells as islated have an extremely lw basal rate f 3-Omethylglucse uptake. This basal rate is biphasic, reflecting a small ppulatin f cells with finite uptake rates and mst cells with rates that are essentially zer (Hawrth et al., 1984). This prperty allwed us t mnitr 3-O-methylglucse uptake cntinually n the same time scale and in the same experiment as we mnitred the nset f cntracture. Figure 6 shws the rime curse f 3-O-methylglucse uptake by cells t which the 3-O-methylglucse was added 30 minutes befre time zer. The subsequent additin f rtenne plus 6 pml FCCP/ mg clearly induced the further uptake f 3-O-methylglucse after a lag perid. Unexpectedly, 3-Omethylglucse uptake was induced in cells withut isprterenl significantly befre it was induced in cells with isprterenl (Fig. 6; Table 1). The nset f cntracture, n the ther hand, ccurred significantly later fr cells withut isprterenl than fr cells with isprterenl (Table I), in agreement with the data in Figure 1. Sme idea f the relative timing f events was gained by cmparing the time at which the inhibitr-stimulated 3-O-methylglucse uptake was half cmplete (ti/2 3-O-methylglucse) t the time at which half f the cells had undergne cntracture (ti/ 2 cntracture). Fr cells withut isprterenl, there was n significant difference between these ti/2 values. Fr cells with isprterenl, 3-O-methylglucse influx appeared t ccur significantly later than the nset f cntracture, by abut nine minutes under these cnditins (Table 1). It thus appears that isprterenl can reduce the impact f glucse influx, nt nly by reducing its extent, but als by delaying its nset with respect t the cntracture event. Discussin Glucse transprt can be induced in whle hearts by uncuplers f xidative phsphrylatin, anxia n <D 100 *~ «s rtenne.+ FCCP FIGURE 6. The effect f isprterenl n the rate f nset f 3-Omethylglucse permeatin. T cells in experimental medium (n glucse) were added ["Q3-0-methylglucse 080 MH 0.3 iici/ml) plus 3 HjO (1 fici/ml) 30 minutes befre time zer. At time zer, isprterenl (1 MM) was added as shwn (filled symbls). Rtenne (3 MM> plus FCCP (6 pml/mg) was added 6 minutes later t (, O) but nt t (A, A). Samples (0.5 ml) were centrifuged at the times shwn (see Methds). In the same experiments, samples were als remved and fixed fr mrphlgical analysis (Table 1). (Mrgan, et al., 1959), r by an increased wrklad (Neely et al., 1967). It is pssible that the mechanism perative in these three cases is ne and the same. The cnditins used here mst clsely apprximate anxia. The signal respnsible fr the inductin f glucse transprt is nt knwn, but appears t arise frm a substrate supply/demand imbalance. The data in Figures 5 and 6, and Table 1, suggest that the signal is given at a time when the cell is nearing the end f its endgenus substrate that is, when the lss f cellular ATP is imminent (Fig. 3). An apparent lack f crrelatin between ATP lss and sugar uptake inductin when glucse is present has TABLE 1 Effect f Isprterenl n the Onset f 3-OMethylglucse Permeability Ispr- + Isprterenl terenl A P ty, cntracture 17.2 ± ± ±1.1 <0.025 (min) t* 3-0-methyl ± 3, ± ± 3.0 <0.05 glucse (min) A 0.7 ± ±4.0 P NS <0.05 Cnditins were as described in the legend t Figure 6. Results are mean ± SD frm three experiments in which bth U (cntracture) and ty, (3-O-methylglucse) were measured. The significance (P) f differences between ty, values (A) was evaluated with the paired /-test.

8 164 been fund in skeletal muscle (Krbl et al., 1977). Hwever, the beneficial effect f the glucse can itself delay the nset f cnrracture when metablic lads are lw, thus bscuring the crrelatin fund withut glucse (Table 1; Fig. 3). The inductin f glucse transprt by insulin is thught t invlve the mvement (recruiting) f prefrmed glucse transprters frm the Glgi fractin t the plasma membrane (Suzuki and Kn, 1980; Cushman and Wardzala, 1980) by an ATPrequiring prcess (Siegel, and Olefsky, 1980). Since glucse transprt cannt be induced by metablic demand beynd that induced by insulin (Mrgan et al., 1959), the inductin f transprt by metablic demand culd well invlve the recruiting f the same pl f glucse transprters. This suggests a pssible mechanism fr the bserved dependence f 3-O-methylglucse transprt rate n metablic demand (Fig. 4B). If the recruiting f transprters by metablic demand takes a certain amunt f time, it culd be prematurely terminated in cells with a high metablic demand by the lss f ATP: the time between the signal t recruit transprters and the lss f ATP culd well be inversely related t the rate f ATP utilizatin, if the signal is itself initiated in sme way by near exhaustin f endgenus substrate. Thus, fr the sake f illustratin, suppse that the signal fr recruitment was given when a cell used up its endgenus glycgen, when its nly remaining (anaerbic) resurces were its ATP plus sme creatine phsphate. The time available fr the ATP-dependent mvement f transprters t the sarclemma wuld be shrt if the cell had a high metablic demand and used up its residual ATP quickly. This wuld result in nly a partial activatin f glucse transprt, the prcess being arrested (see Fig. 4B) by ATP lss (Fig. 3). Fr cells with a lwer metablic demand, the ATP pl wuld be available fr lnger after the initiatin f the recruitment signal, allwing the cmplete mvement f transprters t the sarclemma. The effect f isprterenl n the timing f glucse transprt inductin (Fig. 6; Table 1) appears anmalus in the terms discussed abve, since it appears t ccur after cnrracture (Table 1) when the cells have lst their ATP (Fig. 3). This culd perhaps be explained by the transprters being recruited at the same time relative t cntracture, as in cells nt treated with isprterenl, but being inactivated by phsphrylatin. The activatin bserved (Fig. 6) wuld then be indicative f the rate f transprter dephsphrylatin in cells with very lw ATP, rather than f the rate f transprter recruitment. The metablic cnsequences f rtenne additin fr the cells here are very similar t thse experienced by cells in whle hearts underging high flw anxia. In bth cases, the pssibility f xidative substrate metablism is eliminated, and the cells must resrt t glyclysis r glycgenlysis as a Circulatin Research/Vl. 58, N. 1, January 1986 surce f ATP. In bth cases, the ph f the bathing medium remains near nrmal. In bth cases, the permeability f the cells' membrane t glucse is initially lw in the absence f insulin; this will be true in the whle heart in the fasted cnditin. Thus, it is pssible that the limitatin f glucse transprt inductin by high metablic demand shwn here in islated cells is a significant factr determining the survival f heart cells in viv expsed t high flw anxia. The effect f isprterenl n glucse transprt in viv culd be greater than that bserved here n quiescent cells, since the intrpic effect in viv will increase the metablic demand much mre. This wrk was supprted by a Grant-in-Aid frm the American Heart Assciatin with funds cntributed in part by the Wiscnsin Affiliate, and by Grant HL frm the Natinal Institutes f Health. Address fr reprints: Dr. Rbert A. Hawrth, Department f Surgery, University f Wiscnsin Clinical Science Center, 600 Highland Avenue, Madisn, Wiscnsin Received December 3, 1984; accepted fr publicatin Octber 30, References Cushman SW, Wardzala LJ (1980) Ptential mechanism f insulin actin n glucse transprt in the islated rat adipse cell. J Bil Chem 255: England PJ, Randle PJ (1967) Effectrs f rat-heart hexkinases and the cntrl f rates f glucse phsphrylatin in the perfused rat heart. Bichem J105: Hawrth RA, Hunter DR, Berkff HA (1980) The islatin f Ca 2+ -resistant myytes frm the adult rat. J Ml Cell Cardil 12: Hawrth RA, Hunter DR, Berkff HA (1981) CntTacture in islated adult rat heart cells. Rle f Ca 2+, ATP and cmpartmentatin. Circ Res 49: Hawrth RA, Hunter DR, Berkff HA (1982a) Mechanism f Ca 2+ resistance in adult heart cells islated with trypsin plus Ca 2+. 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9 Hawrth and Berkff/'Cntrl f Sugar Uptake by Metablic Demand 165 tin f glucse transprt and phsphrylatin in the islated, perfused heart f nrmal rats. J Bil Chem 236: Neely JR, Liebermeister H, Mrgan HE (1967) Effect f pressure develpment n membrane transprt f glucse in islated rat heart. Am J Physil 212: Pilch PF, Axelrd JD, Clell J, Czech MP (1981) Unimpaired signal transductin by the adipcyte insulin receptr fllwing its partial prtelytic fragmentatin. J Bil Chem 256: Siegel J, OlefskyJM (1980) Rle f intracellular energy in insulin's ability t activate 3-O-methylglucse transprt by rat adipcytes. Bichemistry 19: Sklff L Reivich M, Kennedy C, DesRsiers MH, Patlak CS, Pertigrew KD, Sakurada O, Shinhara M (1977) The [ 14 C]- dexyglucse methd fr the measurement f lcal cerebral glucse utilizatin: Thery, prcedure, and nrmal values in the cnscius and anesthetized albin rat. J Neurchem 28: Suzuki K, Kn T (1980) Evidence that insulin causes translcarin f glucse transprt activity t the plasma membrane frm an intracellular strage site. Prc Natl Acad Sci USA 77: Takala TES, Hassinen IE (1981) Effect f mechanical wrk lad n the transmural distributin f glucse uptake in the islated perfused rat heart, studied by reginal dexyglucse trapping. Circ Res 49: Wallenstein S, Zucker CL, Fleiss JL (1980) Sme statistical methds useful in circulatin research. Circ Res 47: 1-9 Whitesell RR, Gliemann J (1979) Kinetic parameters f transprt f 3-O-methylglucse and glucse in adipcytes. J Bil Chem 254: Wick AN, Drury DR, Hakada HI, Wlfe JB (1957) Lcalizatin f the primary metablic blck prduced by 2-<Jexyglucse. J Bil Chem 224: INDEX TERMS: Glucse Transprt Cntracture 3-O- Methylglucse 2-Dexyglucse Insulin Dwnladed frm by n January 11, 2019