The effects of insulin on myocardial metabolism and acidosis in normoxia and ischaemia

Biochimica et Biophvsica Acta. 720 (1982) 17-27

17

Elsevier Biomedical Press BBA 11004

T H E EFFECTS OF INSULIN ON MYOCARDIAL METABOLISM AND ACIDOSIS IN

NORMOXIA AND ISCHAEMIA A 31p.NMR STUDY IAN A. BAILEY *, G E O R ( i E K. RADDA, A N N E - M A R I E L, SEYMOUR and STEPHEN R, WILLIAMS **

Department of Biochemistry, UniversiO' of Oxford Sou/h Parks Road, Ox/))rd OXI 3QU (U.K.) (Reccivcd July 8th, 1981 )

Key words: Insulin; Acidosis," Ischemia; Oxidatit:e metabolism; 31p-NMR, (Rat heart)

1. The effect of fnsulin on the perfused rat heart during normoxia and total ischaemia was studied by 31P-NMR. 2. During normoxic perfusion, insulin increased the phosphocreatine to ATP ratio at the expense of Pi, when glucose was the substrate. N o change was observed when acetate was used as the sole substrate. The iutracellular pH (as measured from the position of the 2-deoxyglucose 6-phosphate resonance peak) was unaffected by insulin treatment. 3. Infusion of insulin prior to ischaemia caused an increase in the rate and extent of acidosis during the period of no flow while the rate of ATP depletion was decreased. 4. Freezeclamped studies showed an increase in glycogen levels upon insulin treatment of the glucose perfused rat heart. During ischaemia, a decrease in glycogen content concomitant with an increase in lactate was observed. The accessibility of glycogen to phosphorylase during isehaemia is increased as a result of insulin treatment. The control of glycolysis during ischaemia is discussed with respect to the content and structure of glycogen in heart tissue.

Introduction

Infusion with high concentrations of glucose, insulin and potassium (the G.I.K. system) was suggested some 20 years ago as a means of protecting hearts from ischaemic damage [1]. Whether insulin is beneficial in this system, clinically or experimentally, and how it exerts any beneficial effect remains unclear [2-5]. As glycogen breakdown is vital in maintaining the concentration of ATP in ischaemia in perfused rat hearts subjected

* Present address: ICI Ltd., Pharmaceuticals Division, Mereside Alderley Park, Macclesfield, Cheshire SKI() 4TG, U.K. ** Present address: Kodak Ltd., Research Laboratories, Headstone Drive, Harrow, Middlesex, U.K. 016%4889/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

to total global ischaemia [6], insulin could exert a protective effect by modulating glycogen metabolism. Alternatively, as the hydrolysis of glycolytically-produced ATP results in ischaemic acidosis [6] the insulin-induced increase in glycolytic flux could increase ischaemic acidosis and, thereby, tissue damage. Both ischaemic acidosis and the concentration of ATP can be measured noninvasively in the perfused heart by using 3tp-NMR. In previous experiments [7] we have used the 2-deoxyglucose 6-phosphate accumulated in rat hearts perfused with 2-deoxyglucose to inhibit selectively the activity of phosphorylase b (EC 2.4.1.1) in ischaemia. We have also used the position of its 3~p-NMR resonance to measure intracellular pH. The entry and phosphorylation of radioactively-labelled 2-deoxyglucose has been

18

used widely to measure the rates of glucose transport and phosphorylation by hexokinase (EC 2.7.5.1) in the presence and absence of insulin [8]. Using 3~p-NMR, the accumulation of 2deoxyglucose 6-phosphate can be followed in sequential measurements on the same heart and thus we can differentiate between accumulated 2-deoxyglucose and 2-deoxyglucose 6-phosphate. This is important in analysing whether glucose transport or hexokinase is rate-limiting. In this paper we present 3JP-NMR measurements of the effect of insulin on the intracellular concentrations of phosphocreatine ATP and Pi, on the accumulation of 2-deoxyglucose 6-phosphate and on intracellular pH in normoxia. We also report the effects of the hormone on ATP maintenance and acidosis in total global ischaemia. The observations support the contention that the rate and extent of acidosis and ATP depletion in ischaemia are determined by the availability of myocardial glycogen to glycogen phosphorylase. Methods

Hearts from 280-320g male Wistar rats (Oxfordshire Laboratory Animal Centre Ltd.) were perfused via the aorta [9] as described elsewhere [7]. Krebs-Henseleit bicarbonate buffer containing either 5 mM D-glucose or 5 mM sodium acetate was used. Acetate was used in order to obviate the effects of insulin on glucose transport. Where indicated 1 mM 2-deoxyglucose (Sigma) was added. In phosphate-free perfusions the KH2PO 4 was replaced by an equivalent concentration of KC1. In some experiments a 200 nM solution of 3-times recrystallised bovine insulin (Boots Pure Drug. Co.) in buffer was infused into the buffer in the aortic perfusion line for 1 h at a rate of 0.6 ml ~min ~ to give a concentration at the heart of about 20 nM. This would stimulate glucose transport maximally in the heart. The concentration of insulin in the coronary effluent was measured by radioimmunoassay (insulin R.I.A. kit: Radiochemical Centre, Amersham). The hormone concentration rose to 20 nM during the infusion and fell to 50 pM after washing the system. 31p-NMR measurements were made by applying 45 ° pulses to the sample at 1 or 2 s repetition rates. Spectra were obtained using a 4.2 Tesla

superconducting magnet (Oxford Instruments Ltd.) interfaced with either a Nicolet BNC 12 or 1180 computer. The intracellular pH was calculated from the position of the intracellular P~ or 2deoxyglucose 6-phosphate resonances by reference to standard curves, or by calculation [7]. 5-min spectra (300 transients) were collected before and during insulin infusion. 15 2-min spectra were collected during and 3 before the 30 min total global ischaemia, which was induced by switching off the supply of buffer to the heart (full description of methods in [6]). The concentrations of metabolites were calculated by reference to methylene diphosphonate in 2H20 as a standard, contained in the annulus of the sample probe. In a further series of experiments, hearts were perfused for 1 h in the presence and absence of 50 nM insulin and subjected to total global ischaemia for 5,15 and 30 min before freeze-clamping. The somewhat higher insulin concentration used in these experiments compensates for the greater insulin binding to the particular perfusion apparatus used for these experiments. Part of each heart is extracted in 6% perchloric acid [10] and the concentration of lactate in the neutralised extract was determined fluorimetrically [11]. Samples of frozen tissue were also extracted in 30% KOH and ethanol [12] and their glycogen content determined with phenol and sulphuric acid [13]. A third series of hearts were perfused for l h with and without 50 nM insulin and subjected to 0, 15 and 30 min total global ischaemia. They were then freeze-clamped, extracted in 4.5 ml of 30% KOH, their glycogen precipitated in 50% ethanol [ 13] and suspended in 2 ml water. The accessibility of this glycogen to AMP-activated phosphorylase b which acts only on a-l-4 linked glucose chains was calculated as follows. The total glycogen content was determined as above. The concentration of glucose equivalents produced by the action of phosphorylase b on isolated glycogen was measured by the following modification of the method of Entmann et al. [14]. To each sample of precipitated glycogen was added 2.0 ml distilled water and 0.l-ml aliquots were assayed in 1.8 ml 50 mM triethanolamine-HC1 buffer, pH 7.0, containing 50 mM KC1, 10 mM KHEPO4, 10 mM MgCI 2, 1 mM AMP, 2 m M NADP, 10U phosphoglucomutase (EC 2.5.7.1: Boehringer) and 15U glucose-6-

19 phosphate dehydrogenase (EC 1.1.149: Boehringer). The change in the fluorescence emission intensity of N A D P H was measured following 90 min incubation at 37°C with 1/~M phosphorylase b. The amount of glucose 1-phosphate produced from glycogen was obtained by comparing the fluorescence change with that arising in the assay system from known concentrations of glucose 1phosphate (Sigma) and the concentration of glucose equivalents calculated. The quotient of this value with the total glycogen concentration (both in m g / m l ) taken as a percent is the accessibility of the isolated glycogen to phosphorylase. Purified phosphorylase b was prepared using the method of Fischer and Krebs [15], substituting dithiothreitol for cysteine. Fluorescence measurements were made on a Hitachi MPF-2A spectrofluorimeter using excitation and emission wavelengths of 360 and 460 nm, respectively. Values are quoted thoughout this paper as the mean and standard deviation of the number of replicate measurements unless otherwise indicated.

l,/ '1'

a

b

Results Normoxia

The effect of 15 rain perfusion with insulin on the 3~P-NMR spectrum of a rat heart perfused with buffer containing 5 m M glucose but no Pi is shown in Fig. 1. Insulin increases the phosphocreatine peak height while reducing that of the intracellular Pi to a barely detectable level. The time-course of these events was studied in the presence of buffer Pi by collecting sequential spectra before and after the addition of insulin (Fig. 2). Allowing for the delay before insulin reaches the heart (2 min), the hormone increases the intracellular concentration of phosphocreatine by 12% in 5 rain while leaving the concentration of ATP essentially unaffected (5% increase). The intracellular Pi concentration falls by 10% in the same period. When hearts are perfused with 5 InM sodium acetate, as substrate in the presence of phosphate buffer, the tissue concentrations of phosphocreatine, ATP and Pi are unaltered by the addition of insulin (Table I). In the absence of insulin the p h o s p h o c r e a t i n e / A T P ratio was 3.78-+0.32 (52 determinations on six hearts) with acetate as sub-

Fig. I. The effect of insulin on the 31p-NMR spectrum of a pcrfused rat heart. 31p-NMR spectra of a heart perfused in Krebs-Henscleitbicarbonate buffer containing 5 mM D-glucose but without buffer Pi both before (a) and after 15 min (b) infusion with insulin. Each spectrum is the result of accumulating 300 frce induction decays from 45° pulses applied at 1 s intervals. Assignments: I, intracellular Pi; lI, phosphocreatine; III, T-ATP, fl-ADP; IV, a-ATP, a-ADP; V, fl-ATP,

strate, whereas with glucose the significantly smaller value of 1.99 -+ 0.19 (71 determinations on eight hearts) was obtained ( P < 0 . 0 0 1 ) . In the presence of insulin with glucose as substrate the p h o s p h o c r e a t i n e / A T P ratio is 3.79-+ 0.23 (25 determinations on five hearts). The effect of insulin on the time course of accumulation of 2-deoxyglucose 6-phosphate is shown in Fig. 3. In the absence of insulin, the accumulation of this sugar is linear over 2 h of perfusion under these conditions [7], but these results show that insulin slightly decreases the rate of 2-deoxyglucose 6-phosphate accumulation.

20

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Fig. 2. The effect of insulin on the c o n c e n t r a t i o n of p h o s p h o r u s - c o n t a i n i n g m e t a b o l i t e s in hearts perfused with buffer containing glucose. 20 n M bovine insulin was a d d e d to the aortic perfusion line after 30 min n o r m o x i c perfusion with buffer c o n t a i n i n g 5 m M glucose. Sequential 2 min spectra (120 scans) were o b t a i n e d by a p p l y i n g 45 ° pulses eve W I s over a 0.5 h period to assess the effect of insulin on the heart. The value for the c o n c e n t r a t i o n of p h o s p h o c r e a t i n e , A T P and Pi o b t a i n e d from each s p e c t r u m was percentaged to the m e a n of the six m e a s u r e m e n t s before the action of insulin. The variation u, ith timc of the p e r c e n t a g e of p h o s p h o c r e a t i n e (@), A T P ( © ) and Pi ( A ) are shown. For clarity, d a t a have been s u m m e d over s e q u e n t i a l 4 rain periods, T indicates the b e g i n n i n g of infusion with insulin.

The effect of insulin on the intracellular pH of hearts perfused with buffer containing 5 mM glucose can be measured using the P~ resonance position. However, because the intracellular P~ concentration falls in the presence of insulin, the resultant P~ resonance is sometimes difficult to differentiate from that arising from buffer Pi. However, careful analysis shows that the intracellular pH does not change under the influence of insulin. This problem of resolving signal from internal and external compartments does not arise with the 2-deoxyglucose 6-phosphate resonance as this sugar is located exclusively in the cytoplasm [7]. When this resonance is used to measure the effect of insulin on hearts perfused with 5 mM glucose and l mM 2-deoxyglucose, no effect of insulin on the myocardial pH. is observed (Table II). The pH measured, however, is slightly lower than normal as the phosphorylation of 2deoxyglucose and its subsequent dissociation produces protons [7]. Ischaemia The rates of ischaemic acidosis and ATP depletion in global ischaemia of hearts perfused for 1 h in the presence of insulin with 5 mM glucose as substrate are shown in Fig. 4. The intracellular pH

TABLE I THE EFFECT OF INSULIN ON THE CONCENTRATIONS PERFUSED WITH ACETATE

OF PHOSPHORUS-CONTAINING

M E T A B O L 1 T E S IN H E A R T S

3 z P - N M R spectra were o b t a i n e d from hearts perfused with n o r m a l Krebs-Henseleit b i c a r b o n a t e buffer c o n t a i n i n g 5 m M sodium acetate as s u b s t r a t e b y a c c u m u l a t i o n of 300 transients at 2 s intervals. F r o m these the c o n c e n t r a t i o n s of p h o s p h o c r e a t i n e , ATP and P, ,.','ere m e a s u r e d and p e r c e n t a g e d relative to the m e a n values of three m e a s u r e m e n t s made before infusion with 20 nM bovine insulin. M e a n " S.D. of five hearts are q u o t e d in each case. N o significant change is observed when a p p l y i n g the Students t-test to ,,alues i n d i c a t e d by an asterisk. ( P > 0 . I). T i m e relative to start of insulin infusion (min) " 25 15 5 5 15 25 35 45 55

Relative c o n c e n t r a t i o n (%) Phosphocreatine

ATP

Pi

104,8 + 100.2 ~ 95,9 + 92.9 + 102.5 + 99.4 + *103.4 + 112.495.0 ~

99.8+2.1 102.1+1.7 96.6~3.3 98.449.1 98.7+3.9 97.1 +5.8 98.5~5.9 100.g~6.4 96.7 + 2.2

97.6 +10.7 97.9 * 6.0 104.0" 8.7 110.0 ~ 4.S 108.4 ÷ 11.0 1 0 6 . 5 - 13.5 *113.7 * 3.8 * 99.1 ~ 15.8 *111.8" 10.5

6.2 4.4 5.9 9.7 7.3 12.5 II.I 8.7 5.1

21

T A B L E II V A R I A T I O N OF I N T R A C E L L U L A R pH ON T H E A D D ITION OF INSULIN, MEASURED USING 2DEOXYGLUCOSE 6-PHOSPHATE

40(

Hearts were perfused for I h with buffer containing I mM 2-deoxyglucose and 5 mM glucose. 200 nM insulin was then infused into the aortic perfusion line at a rate of 0.6 ml 1.rain I for a further hour. 10-rain 31p-NMR spectra (300 scans: 45 ° pulses every 2 S) were taken throughout this period, pH was calculated from the resonance position of 2-deoxyglucose 6phosphate as described by Bailey et al. [7]. The time quoted is that midway in the aecunm|ation. The mean ~ S.D. of pH measurements on six hearts are shown.

3o~

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Time (min) relative to the addition of insulin

o >,

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(rain).

Fig. 3. The effect of insulin on 2-deoxyglucose 6-phosphate accumulation in the rat heart. The accumulation of 2deoxyglucosc 6-phosphate before and during infusion with 20 nM bovine insulin is shown as the mean ± S.D. of five hearts. Each point represents the sugar phosphate concentration measured from the 31P-NMR spectrum obtained by the accumulation of 300 transients at I s intervals. ]" indicates the beginning of infusion with insulin.

falls to 5.65-+0.03 (five hearts) after 29 min ischaemia. The rate of change in ATP content is slowed down in the presence of insulin q/2 is 16 rain in the presence of insulin (see Fig. 4b), whereas in the control situation tt/2 is 9 min (see Ref. 6). If 1 mM 2-deoxyglucose was added to the perfusion medium for the duration of the insulin treatment, the intracellular pH fell to 6.1 ± 0.1 (five hearts) after 23 min of ischaemia while ATP concentration was observable for 24 rain (Fig. 5). Changing the exogenous substrate to 5 mM sodium acetate had the effect of limiting the ischaemic pH at 29 min to 6.4-+ 0.2 (five hearts) (Fig. 6) while the ATP concentration is maintained for some 15 min (data not shown). Table III shows that the production of lactate

-22.5 17.5 - 12.5 - 7.5 2.5 + 2.5 7.5 12.5 17.5 22.5 27.5 32,5 37.5 42.5 47.5 52.5

Intraccllular pH from 2-deoxyglucose 6-phosphate resonance 7.01 +0.02 7.00 + 0.05 7.01 +0.03 7.01+0.02 7.00 ~0,01 6.99+0.03 6.98+0.01 6.99 " 0 . 0 2 6.99 ± 0,02 7.00 + 0.0 I 7.00 ~0.02 7.00 + 0.02 7.00±0,02 7,00 + 0,02 7.00 + 0,02 7.00 ~ 0.02

in the presence of insulin continues over the 30 min of total global ischaemia, while that in its absence does not increase after 15 min of ischaemia. The rise in lactate levels parallels the decrease in myocardial glycogen in general. The accessibility to phosphorylase b of the glycogen remaining in total global ischaemia is shown in Table IV. The data show that as the duration of ischaemia increases, a smaller percentage of the remaining glycogen is accessible to phosphorylase b whether or not insulin is present. Pre-infusion with insulin increases the amount of accessible glycogen at the start of the ischaemic period (P <

0.001).

22

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310

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T i m e (rain)

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Fig. 4. Acidosis and ATP depletion in ischaemia of insulin-treated hearts perfused with glucose buffer. Hearts were perfused for 1 h with buffer containing 5 mM D-glucose in the presence of 20 nM insulin and then subjected to total global ischaemia. 2 rain spectra were obtained throughout the ischaemic period by the accumulation of 120 free induction decays generated by 45 ° pulses cver~ I s. ]'he metabolite concentration and resonance positions were corrected relative to the methylene diphosphonate external standard. Intracellular pH (panel a) was calculated from the Pi resonance and the ATP content (panel b) was percentaged to the mean of six control spectra obtained immediately before ischaemia. Mean ± S.D. of six hearts are shown in each case.

TABLE III VARIATION IN THE C O N C E N T R A T I O N OF LACTATE AND G L Y C O G E N D U R I N G TOTAL GLOBAL ISCHAEMIA: THE E F F E C T OF PREPERFUSION WITH I N S U L I N Hearts were perfused for I h in the presence and absence of 50 nM bovine insulin and then freeze-clamped after 0, 15 and 30 min total global ischaemia. The glycogen and lactate concentrations of portions of each heart were determined as described in Methods. Mean ± S.D. of five hearts are shown. The probability of increased lactate production or increased glycogen utilisation during the last 15 rain of ischaemia was tested using the Students t-test.

Control

Plus insulin

~' 0.1 > P>0.05,

Duration of ischaemia (rain)

Lactate concentration mg-(g wet wt.) i

Glycogen concentration mg.(g wet w t , ) - i

0 15 30

0.093 -+ 0.034 0.481 -+ 0.093 0.317-+0.124

1.27-+ 0.21 0.52 ± 0.19 0.34±0.19

0 [5 30

0.075±0.054 0.360 + 0.088 0.809 ± 0.155

2.67--+0. t4 1.96 -+ 0.72 1.33 ± 0.43 a

23

b

100

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Time (rain)

Fig. 5. Acidosis and ATP maintenance in total global ischaemia of hearts perfused with insulin, glucose and deoxyglucose. Hearts were perfused with buffer containing 1 m M 2-deoxyglucose and 5 m M D-glucose for 2 h. During the second hour, 200 nM,insulin was infused into the aortic perfusion line at a rate of 0.6 ml I.min'l to stimulate the heart maximally. The heart was then subjected to total global ischaemia and 15 2-min 31p N M R spectra (120 45" pulses at I s intervals) were obtained over a 30 rain period. The intracellular p H (panel a) and the percentage of ATP maintained (panel b) were calculated as for Fig, 4. Mean m S.D. of six hearts are shown.

Discussion

Normoxie phosphorylations

6.6

I

T o_

II

6.2

I

0

On stimulation with insulin, an increase in the concentration of phosphocreatine at the expense of intracellular Pi is observed while there is little change in the concentration cf A T E This effect is abolished when acetate is used as a sole exogenous substrate. The effect must therefore be dependent upon the effect of insulin on glucose transport and glycolysis. Kobayashi and Neely [16] have shown that tricarboxylic acid cycle activity is substrate-

III ITI]I

I

I

I

10

20

30

Time (rain)

Fig. 6, Ischaemic acidosis in the rat heart perfused with acetate buffer. Hearts were perfused with insulin for l h in buffer

containing 5 m M sodium acetate and subjected to total global ischaemia. 2 rain 31p-NMR spectra (120 transients arising from 45 ° pulses every l s) were obtained throughout 30 min of ischaemia. The intracellular pH measured from the Pi resonance is shown as the mean-+S.D, of six hearts.

24 TABLE IV THE ACCESSIBILITY OF MYOCARDIAL G L Y C O G E N TO PHOSPHORYLASE h IN ISCHAEMIA Hearts were perfused in glucose buffer for I h in the presence and absence of 50 nM insulin and freeze-clamped after 0, 15 and 30 min total global ischaemia. The glycogen from each was precipitated in 50~ ethanol after alkaline extraction and resuspended in 2 ml water. The concentration of glycogen in m g / m l was then determined in triplicate by the phenol/sulphuric acid method. The concentration of glucose equivalents in m g / m l produced by' the action of glycogen phosphorylase was determined fluorimetrically, again in triplicate. The percentage accessibility quoted represents the percentage of the mean fluorimetric to the mean chemical determination for each heart. Values are the m e a n = S.D. of five hearts in each case. Presence of 50 nM insulin

+ +

Duration of ischaemia (rain)

Percentage accessibilit}

0 15 30

52.7 ~ 7.0 25.6" 6.7 15.9 ! 11.5

0 15 30

87.7 ~ 7.4 56.7 + 13.1 48.3 + 18.7

glucose 6-phosphate [18] may account for this observation. The decrease in the rate of 2deoxyglucose 6-phosphate accumulation may indicate an increased rate of utilization. A small quantity of 2-deoxyglucose 6-phosphate can be metabolised either by oxidation involving glucose6-phosphate dehydrogenase [19] or by deposition as glycogen [20]. Insulin may stimulate both these processes. However, given the sensitivity of this preparation of insulin (Figs. 1 and 2) and the known stimulation of 2-deoxyglucose uptake by insulin [8], there is likely to be a considerable build-up of free 2-deoxyglucose unlike the stituation in the absence of insulin [7]. Under the conditions of insulin stimulation, the rate of accumulation of 2-deoxyglucose 6-phosphate does not correspond to the rate of glucose transport [21]. Hexokinase becomes the rate-limiting step. These events occur within several minutes of insulin treatment (Fig. 4). Thus, measuring the rate of insulin-stimulated glucose transport using methods based upon the accumulation appreciable amounts of labelled analogues of glucose may be invalid.

Normoxic pH limited in the heart, at high workloads, when glucose is the sole substrate. This results in low levels of high energy phosphates and acetyl-CoA; if this is so in our case, insulin would stimulate both glucose transport and pyruvate dehydrogenase and will increase substrate delivery to the mitochondria. The increase in the phosphocreatine to ATP ratio, which we observe may reflect these phenomena. This is further substantiated by the experiments using acetate as sole substrate. The phosphocreatine to ATP ratio is higher when using 5 mM acetate as substrate rather than 5 mM glucose, conditions in which tricarboxylic acid cycle activity and oxidative phosphorylation are increased [ 17]. In the presence of buffer phosphate, the intracellular P~ decreases less dramatically than in phosphate free perfusion (Fig. 2) as it is replenished by the entry of P~ from the extracellular pool. The increased rate of sugar transport caused by insulin is not reflected in an increased rate of 2-deoxyglucose 6-phosphate accumulation (Fig. 4). The non-competitive inhibition of hexokinase by

Unlike in frog muscle, [22-24] we find no evidence for an increase in intracellular pH in the presence of insulin in our preparation. As our N M R measurements are made at higher fields, than that used by Gupta and Moore [24] to observe a pH increase of 0.10-+ 0.03, we should see such changes with greater ease as an equivalent p H is spread over a greater number of data points in our N M R spectra. It is not surprising that poikilothermic amphibian skeletal muscle and homeothermic mammalian cardiac muscle behave differently. It may be that the amphibian is simply less efficient in pH homeostasis than the mammal. It may be that poikilothermic skeletal muscle requires much greater surges of glycolytic flux to changes in its environment than does homeothermic cardiac muscle. Indeed, it would be surprising if mammalian cardiac muscle, which regulates so carefully its ionic balance and enzymic activity during the contractile cycle, should permit major alterations in intracellular pH and so jeopardise the control of these processes in any but the most extreme conditions, e.g., myocardial ischaemia.

25 Whatever the species differences, the absence of a change in pH in rat heart muscle would suggest that changes in phosphofructokinase activity with pH do not constitute a major mechanism of glycolytic control in this tissue in normoxia.

Ischaemic acidosis Garlick et al. [6] have demonstrated that the extent of acidosis in the perfused heart is proportional to the amount of glycogen degraded in ischaemia, suggesting that the major source of protons is the hydrolysis of glycolytically-produced ATP. So, in glycogen-depleted hearts, ATP is maintained for only 8 min rather than the usual 15 min and the intracellular pH falls to 6.8 rather than 6.2. Similarly, inhibiting phosphorylase b with 2-deoxyglucose 6-phosphate results in total ATP depletion in 11 min and a final pH of 6.4 [7]. Pre-perfusion for 1 h with a maximallystimulating concentration of insulin in buffer containing glucose results in a decrease in the rate of ATP depletion global ischaemia for some 30 rain (Fig. 5b). This, combined with the increased rate and extent of acidosis simultaneously observed (Fig. 5a), means that both anaerobic ATP production and utilization are increased by insulin. This must be caused by an increased glycolytic flux. This is confirmed by the observation of increased lactate production thoughout the ischaemic period after preperfusion with insulin (Table III). The continued breakdown of glycogen and production of lactate during 30 rain of ischaemia in the presence of insulin indicates that glycolysis continues thoughout this period. The further decrease in pH in the presence of insulin must reflect this continued glycogenolysis generating ATP. Though insulin increases the rate of glucose transport, the use of exogenous glucose cannot account for the observed increase in acidosis in ischaemia. Following Garlick [25], we calculate that with our size of sample compartment containing 5 mM glucose, a maximum of 0.05 pH unit could arise fom the complete utilization of the glucose in the chamber, assuming it were all available to the heart in the no-flow situation. That the substrate for glycolysis is glycogen is attested by the greater amount of and fall in myocardial glycogen in ischaemic hearts preperfused with insulin (Table III).

The extent of ischaemic acidosis and the rate of ATP depletion when buffer containing acetate is used are similar to those observed in the absence of insulin using glucose buffer (Fig. 6 cf. Ref. 6). This is presumably because the effect of the hormone on metabolism is dependent on glucose transport. The small reduction in acidosis and ATP maintenance with acetate buffer is probably the result of a small amount of normoxic glycogen breakdown and residual glycolytic activity. Minor changes in glycogen levels have been observed with normal acetate perfusion [26]. Evidence that insulin exerts its effect in ischaemia via glucose transport increasing the amount of glycogen stored is provided by this experiment and by the direct relationship observed between lactate production and glycogen breakdown in ischaemia (Table III and Ref. 6). These experiments show that it is possible to alter relatively easily the amount of glycogen stored in the heart and thereby to affect differentially the pattern of acidosis and ATP maintenance in ischaemia. Acidosis and ATP depletion have both been suggested as mechanisms of myocardial damage in infarction. We are currently examining the possibility that modulating glycogen metabolism could protect the heart from ischaemic damage [281.

Control of ischaemic glycogen metabolism In the presence of insulin, glycolysis is maintained despite a decrease in intracellular pH to 5.7. In the control, (untreated) situation as described [6] the pH is decreased to 6.2, 30 min after total global ischaemia. Control of glycolysis was often thought to occur at the level of phosphofructokinase-inhibition of the enzyme resulting from the extent of acidosis in ischaemia [29]. However, in vitro studies of phosphofructokinase, using physiological concentrations of ATP and Pi have shown that activity can be maintained at pH values as low as 6.0 [30]. Thus, phosphofructokinase inactivation cannot be responsible for the cessation of glycolysis in ischaemia, pH 6.2. Data from Neely and coworkers [31] suggest that the reaction catalysed by glyceraldehyde- 3-phosphate dehydrogenase becomes rate-limiting during ischaemia owing to the increased concentrations of NAD + and lactate. In

26

view of the results obtained in this study, this explanation could not account for the decrease in glycolysis in the control situation. Lactate concentrations are higher after 15 rain ischemia in insulin pre-perfused hearts than those observed after 30 min ischaemia in the control (Table IV). Thus, it is unlikely that glycogen degradation becomes rate-linmea at pH 6.2 in the absence of insulin as a result of enzymic inactivation at that pH. After 30 min ischaemia, control hearts have low glycogen levels and reduced phosphorylase accessibility to its substrate (Table IV). This can be interpreted in two ways. The glycogen concentration is sufficiently low to cause disruption of the glycogen particle and associated enzyme system. thereby removing the favoured accessibility to phosphorylase. Entman et al. [32] have demonstrated such a dissociation at low glycogen levels. Alternatively, the glycogen remaining after 30 min ischaemia may contain insufficient a - l - 4 linkages, preventing breakdown of glycogen by phosphorylase. We cannot differentiate between these possibilities. However, ideas about the nature of glycolytic inhibition during ischaemia should be reconsidered. This study does, however, provide some evidence for control of ischaemic glycolysis through the tissue glycogen content. Insulin increases both the amount of tissue glycogen (Table IV) and the accessibility of the substrate. This is best explained by insulin increasing the rate of synthesis of a - l - 4 linkages by its action on glycogen synthase [33] while leaving unaffected the activity of the branching enzyme. This is consistent with the reported insensitivity of this enzyme to metabolic effectors. We have shown that it is possible to alter the number of c~-1-4 linkages in the glycogen stored in the heart. Such changes in glycogen content and accessibility to phosphorylase would alter both the extent of ATP maintenance and acidosis in ischaemia and may provide another means of reducing damage occurring during infarction. Acknowledgements The authors would like to thank the Science Research Council, the National Institutes of Health

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