The Effect of Insulin and Growth Hormone on the Flux of Tracer from Labelled Lactate in Perfused Rat Heart

Eur. J. Biochem. 36,369-379 (1973)

The Effect of Insulin and Growth Hormone on the Flux of Tracer from Labelled Lactate in Perfused Rat Heart

John MOWBRAY and James H. OTTAWAY Biochemistry Department, Univemity Medical School, Edinburgh

(Received February 1, 1973)

1. ~[2-14C]Lactate tracer was added at 30 min to the perfused rat-heart preparation described in the previous paper. The specific activities of perfusate and tissue lactate and pyruvate were measured over the period up to 60 min.

2. Perfusate and heart lactate radioactivity equilibrated rapidly : not so perfusate and heart pyruvate.

3. No pyruvate was lost from the tissue: the sole precursor of perfusate pyruvate was lactate. In the presence of insulin all pyruvate taken up from perfusate was obligatorily reduced to lactate.

4. A proportion of pyruvate in the heart did not become labelled and there is good reason to believe this is not an artifact.

5. Rate parameters at different steps in pyruvate metabolism have been determined by computer modelling. Both hormones modify these parameters : in particular pyruvate dehydrogenase appears to undergo rapid 7-fold activation.

6. The rate of exchange of label between lactate and intracellular labelled pyruvate was used to calculate the [NAD]/[NADH] ratios in the exchange compartment and in mitochondria. Both hormones reduced the ratio about 5 times by comparison with control. The results emphasise the disparity between perfusate lactate/pyruvate ratio and the redox potential of cardiac cytoplasm.

7. The data strongly support the compartmentization in more than one distinct cytoplasmic space of some glycolytic enzymes in cardiac tissue. The relationship of these “compartments?? to heart morphology is briefly discussed.

8. Evidence is presented that growth hormone exerts a much greater rapid “insulin-like” effect than has hitherto been suspected.

It is possible by the use of tracer to obtain a great deal of information about the organisation of and the rate parameters of enzymes affecting pyruvate metabolism in the intact beating rat heart (see also the preceding paper [l]). Steady state or, in the case of pyruvate, reproducible non- steady state conditions have been found and the material fluxes measured [l]. Although in reversibly interconnected compartmental systems it does not matter which compartment is initially labelled [2], perfusate lactate was chosen because of its size and constancy [I].

In this study of the kinetics of tracer distribution, alanine has been neglected, on the principle of perturbation [2] since its known rate of incorpora- tion in protein is very low [3] and its accumulation

Enzymes. Lactate dehydrogenase (EC 1.1.1.27); pyruvate dehydrogenase is complex of pyruvate decarboxylase (EC 4.1.1.1); lipoate acetyl transferase (EC 2.3.1.12); lipo- amide dehydrogenase (EC 1.6.4.3); pyruvate kinase (EC 2.7.1.40).

in perfusate was very small. The specific activities of alanine glutamate and some citric-acid-cycle intermediates were, however, always measured.

MATERIALS AND METHODS

Perf usion In these experiments pyruvate was added to the

perfusion system a t zero time so that the peak of perfusate pyruvate accumulation was before 30 min thus making it possible to describe the decline of pyruvate as a simple mathematical expression : moreover it ensured that carbohydrate oxidation was at a maximum [I]. L( +)-[2-14C]Lactate (5.8 mM, 76200 dis. x min-l x pmol-l) was injected by micro- meter syringe at 30 rnin to produce a lactate specific activity in the perfusate of 2000 dis. x min-I x pmol-1 At 29 min after the start of the perfusion a sample was withdrawn to permit analysis of glucose, lactate and pyruvate concentrations. Samples were taken

370 Effects of Hormones on Pyruvate Flux in Heart Eur. J. Biochem.

from the perfusate at 35, 40, 50 and 60min. At 60min the heart was frozen with Wollenberger tongs.

Radiochemicals L( +)-[2-14C]Lactate was prepared by enzymic

reduction of 0.1-mCi batches of sodium [2-W]- pyruvate (Radiochemical Centre, Amersham) con- taining 10 pmol carrier sodium pyruvate. The lactate dehydrogenase was denatured with trichloroacetic acid, 20 pmol carrier lactate added and the protein spun down. 50mg sodium acetate and 0.5g semi- carbazide, hydrochloride were added to remove unreacted carbonyl compounds and after 30 min the trichloroacetic acid was removed by washing with chloroform. Lactic acid was removed from the acidified solution in a N, stream, water was added and lactides hydrolysed by boiling before neutraliz- ing with NaOH. The recovery with fresh (see [4]) pyruvrtte was almost complete. The material assayed as L( +)-lactate with lactate dehydrogenase and NAD, and gave a single peak which co-chromato- graphed with authentic lactic acid on silicic acid [5].

Pyruvate specific activity was estimated by the method of Mowbray and Ottaway [6]. Lactate specific activity was estimated either directly after chromatography on Dowex-50 [7] or by difference from pyruvate from the total radioactivity in a perfusate sample acidified to pH 4 and made C02- free by evacuation at a water pump for 10min. These methods agreed very closely [B]. The scintil- lator was a dioxane-based “Scinstant” (Nuclear Enterprises Ltd., Edinburgh) and the spectrometer a Nuclear Enterprises NE 8304. With a background rate of about 1 count/s the standard deviation of the counts was always less than 2.3O/,. Quench correction was by the channels ratio method [9].

RESULTS Redistribution of Label in Lactate and Pyruvate Although believed unlikely [l], accumulation of

double label in lactate or pyruvate would seriously distort the interpretation of specific activity data. Lactate and pyruvate were thus purified [5] from 60-min perfusate and subjected to stepwise degrada- tion [lo]. In all cases the radioactivity in carbon atoms 1 and 3 was negligible.

The Equivalence of the Labelled Tissue and Perfusate Pools of Metabolite

If transfer across the cell membrane of lactate and pyruvate was rapid compared to metabolism then the specific activities of these compounds in perfusate might reflect the internal specific activities very closely. The specific activities of lactate isolated

[5] from perfusate and extract (not corrected for perfusate contamination) were indistinguishable. Pyruvate specific activities, on the other hand, were very different: for example in 5 non-hormone- treated hearts the extract specific activity was 100 & 14 dis. x s-l x pmol-1 x g heart-l and the perfusate value was 491 & 11 dis. x s-l x pmol-l x g heart-l.

The possibility that the extract specific activity was artifactually low because of breakdown of unlabeled compound in the preparation of the extract was tested by adding oxaloacetate and phosphoenolpyruvate to frozen heart powder. The recovery of both in neutralised perchloric acid extract was about 99 O/w Phosphoenolpyruvate was not converted to methyl quinoxalone [6] and oxaloacetate was routinely removed by malate dehydrogenase. Oxaloacetate, moreover, was relatively highly labelled.

There remains the possibility that substantial quantities of phosphoenolpyruvate are transformed into pyruvate during the freeze-stop proce- dure which takes about 150 ms [ll]. At the maximum rate of lactate production [12] working anoxic hearts could produce 0.1 pmol pyruvate in this time. Against this, our nonworking nonanoxic hearts only produce 1.2 pmol pyruvate per minute [l]. Further since a t maximum lactate production the pyruvate pool size does not increase [12] an appreciable decrease in lactate specific activity should also have occurred. Finally the size of the discrepancy between the pyruvate specific activity of heart and perfusate ought to be proportional to tissue pyruvate pool size which is not a t all the case (see below).

The Distribution of Label after 30-min Perfusion with [2-14CJLmtate

The specific activity values obtained in the three series of experiments are given in Table 1. For reasons of time it was not possible to estimate the perfusate pyruvafe specific activity in all samples, so measurements were made a t 40 and 60 min only. When the shape of the perfusate pyruvate specific activity was approximately established, it was possible to interpolate values for pyruvate specific activities at 35 and 50 min. There was little error involved in this, because the pyruvate curve could not vary greatly and the correction to lactate involved was only about 5 O / , of the total radioactivity in lactate. However as explained in the following theoretical paper [13], in the actual optimisation which produced the estimates of the rate constants, total amounts in the tracer and chemical pools respectively were used, so that no question of inter- polation arose. In this paper specific activities are used in general, as the argument is easier to follow.

Vol. 36, No. 2,1973 J. MOWBRAY and J. H. OTTAWAY 371

Table 1. Specific activities of lactate and pymuate during perfusion with[2-14C]luctate All values (f S.E.M.) have been corrected to an initial lactate specific activity of 1000 dis. x s-1 x pmol-1~ g heart-1. The values at 35 and 50 min were corrected using interpolated pyruvate specific activities (see text). Insulin, where given was 100 mU/ml

and growth hormone was 4 wg/ml

Control (8 hearts) Insulin (9 hearts) Growth hormone (6 hearts)

Time Perfusate Pyruvate Perfusate Heart Perfusate Heart Lactate Pyruvatc Heart Lactate Pyruvate 'fluvat' Lactate Pyruvate 'yruvate

dis. x s--l x I*mol-l x g-'

30 1000 0 0 1000 0 0 1000 0 0 35 934 f 50 859 f 30 907 f 53 40 8 1 6 f 5 2 238 f 12 839 f 30 479 f 4 9 828 & 46 513 & 37 50 691 f 49 807 f 34 703 f 33 60 5 0 8 f 3 5 4 5 6 f 3 0 136f15 7 2 1 f 4 1 7 3 3 f 4 1 9 2 & 4 8 6 0 9 f 2 9 6 4 2 f 3 5 7 4 f 2 4

Table 2. Concentrationa of lactate and pymvate during perfusion with [2-14C]lactate The perfusate values are pmol x ml-l x g heart-l & S.E.M.: heart values are pmol/g f S.E.M. The data refer to the same hearts as in Table 1. In perfusate the zero-time lactate concentrations were 0.60, 2.12 and 0.72 mM/g and the zero time pyruvate concentrations were 0.1, 0.2 and 0.1 mM/g for control, insulin and growth hormone hearts, respectively. The average perfusate volume was 48.42 ml. The 30-min lactate upon which the initial lactate specific activity depends, was interpolated by linear

regression on 29-60 min values

Control Insulin Growth Hormone Time

Lactate Pymvate Lactate Pyruvate Lactate Pyruvate

min

Perfusate 30 0.61 f 0.051 0.217 f 0.021 2.12 f 0.026 0.219 f 0.017 0.75 f 0.025 0.136 f 0.018 35 0.61 f 0.046 0.178 & 0.013 2.11 f 0.034 0.197 f 0.016 0.725 & 0.021 0.127 f 0.017 40 0.64 f 0.047 0.148 f 0.011 2.10 f 0.027 0.170 f 0.016 0.735 f 0.036 0.119 & 0.012 50 0.65 f 0.052 0.094 f 0.012 2.03 f 0.037 0.125 f 0.017 0.72 f 0.023 0.082 f 0.010 60 0.68 f 0.051 0.057 f 0.013 2.01 f 0.037 0.076 f 0.011 0.675 & 0.023 0.066 f 0.009

w o l x ml-' x g-'

I*mol/g

Heart 60 0.63 f 0.064 0.093 f 0.016 1.67 f 0.25 0.049 f 0.003 0.64 f 0.11 0.034 f 0.003

Table 2 gives the values for the concentrations of lactate and pyruvate during the 30min of the tracer experiments.

Selection of a Model to Fit the Tracer Fluxes

The modelling proceeded in stages, in the first of which the extracellular pyruvate pool was neglected. Since lactate and intracellular pyruvate specific activities are very different the simplest representation is shown in Model A.

Model A. The Exchange of Lactate and Intracellulur Pyruvate

from glucose

If r3 = r4 (pmol x min-l x g-l) were set equal to the total in0ux of unlabelled pyruvate, it was possible using an analogue computer to find an empirical value of rl = r2 which would permit the decay curve for lactate specific activity to be super- imposed on the experimental one. The calculated minimum specific activity in the pyruvate pool necessary to remove the 14C observed to leave the lactate pool was, however, 2, 4.5 and 8 times the 60-min experimental values in the control, insulin- treated and growth-hormone-treated hearts respectively. It is clear, therefore, that a substantial fraction of the intracellular pyruvate must remain unlabelled (or virtually so), and it cannot be taken for granted that all the newly formed pyruvate mixes with the highly labelled pool of intracellular pyruvate .

The next obvious possibility was that the highly labelled intracellular pyruvate pool and the perfusate pyruvate pool were identical or a t least closely related with a rapid exchange rate so that they could be lumped together (Model B).

372 Effects of Hormones on Pyruvate Flux in Heart Eur. J. Bioehem.

Model B. Perfusate Pyruvate as the Highly Labelled Intracellular-Pyruvate Pool

Glucose - 1.26 -+ Tricar-

Labelled pyruvate

Lactate - r 2 3 in heart

perfusate

boxylic acid cycle

Results from simulating Model B with growth hormone data are given but the conclusions apply equally to the other experiments. The concentration of pyruvate in perfusate can be represented very well by a single exponential (Annex Fig.l) over the period of the tracer experiment and perfusate [pyruvate] = 0.22 - e-0.037t was used. Fig. 1 shows that it was impossible to get this model to fit simultaneously the 40- and 60-min experimental results for the specific activity of perfusate pyruvate. The fundamental difficulty is that the rapid turnover of label in the large lactate pool imposes a “forcing function” on the label in the pyruvate pool through which it is lost to the system, that ensures that well before the end of the experiment the two curves, for lactate and pyruvate, must run parallel to one another. Such a curve cannot fit the observed perfusate pyruvate specific activity. Also the highly labelled intracellular pyruvate cannot be lumped with perfusate pyruvate which must be a mammillary pool [2].

If pyruvate, P, in the perfusate of volume V , is produced a t a constant rate, R, from some precursor in the heart and is removed a t a rate proportional to its concentration, one may write

d [PI 7- = R - k[P]. d t

The solution to this equation is

where [PI, is the zero-time concentration of pyruvate. This predicts that a t d t after a certain time, the pyruvate concentration will asymptote to an equilibrium concentration Rlk. A number of variants of Equation (1) may be written, but all predict an ultimate equilibrium. Since, instead, the pyruvate concentration rises to a maximum and then falls, either R or k in Equation (1) must vary with time.

The possibility that k (the efflux rate constant) should increase with time could be ruled out by considering the tracer data. It would mean that the fractional turnover rate o f the perfusate pyruvate

Perfusion t ime (rnin)

Fig. 1. Analog c m p t e r simulation of Model B (see text). The symbols represent the mean specific activity i. S.E.M. of lactate (0) and perfusate pyruvate ( 0 ) from the control hearts. The lines represent two computed lactate (identical) and perfusate pyruvate times courses. The figure shows that although parameters for this model can be found to fit the lactate values it is not possible to fit both pyruvate points

simultaneously

pool would increase with time, bringing the specific activity of that pyruvate sooner and sooner parallel to that of the lactate. It has already been seen that this is not in accord with the facts, and it is concluded that R (the rate of influx of pyruvate into the perfusate), decreases with time.

It should be stressed that we have no information about the reason for this decrease, and were therefore compelled to represent it by an empirical expression. The only proviso must be that R never becomes negative. Since an exponential decay of the pool itself, governed a t this time largely by the be- haviour of R, represented the data quite well, we were content to represent R by the expression R = R * e-At. In aome optimisation attempts this was replaced by the expression R = R [l - tanh (At)], since it is also impossible for this function to become negative, and the slope of (1 - tanh x) is more linear for small x than e--5. However, the fit to the experimental data was not so good.

About 15 variants on the basic model deduced from the considerations outlined above were tested for fit to the experimental data by the optimisation procedures described by in the following paper [13]; both tracer and chemical data were fitted to each model. These variants will not be described in detail. Two general points may be made. First, in view of the recent interest in the possibility that pyruvate dehydrogenase is a control point in pyruvate metabolism [14,15], particular attention was paid to the possibility that the “rate constant’’ for pyruvate efflux from the system varies with time, or alternatively that pyruvate efflux proceeds at a constant rate. The latter possibility implied that the rate of formation of pyruvate from phospho-

Vo1.38, No.2,1973 J. MOWBRAY and J. H. OTTAWAY 373

enolpyruvate varies with pyruvate concentration, but as the concentrations of reactants and products of the pyruvate kinase reaction are far from equilibrium [ 161 this is not outside the bounds of possibility. In this and all other cases in which rate constants were made time-dependent, it was however found that simple dependence of the fluxes of all substrates on their concentrations, with the single exception of the pyruvate efflux to the perfusate, gave a much better fit to all the data.

The second point is that it was impossible to fit the perfusate pyruvate specific activity curve precisely to the data if this pyruvate were made to come directly from the obvious precursor, the intracellular pyruvate (Annex Model C; Annex Fig. 2).

Moreover measurements of perfusate pyruvate specific activity after perfusion with [l*C]lactate a t zero time in the absence of added pyruvate confirm the rejection of Model C. The specific activities of the perfusate pyruvate a t 5 min and 20 min were not different from those of lactate a t these times. This is not possible unless lactate is the direct precursor of perfusate pyruvate (Model D).

Mode l D

Unlabelled pyruvate 1 i;i:rboxylic

Glucose

P 4 1-1 --I--

R2 -- Lactate pyruvate in - heart __

/ \ \ * - A t R6 RT

R3 - 1 /

in perfusate

The least satisfactory element of the fitting in the variants of Model D was the shape of the lactate specific activity curve in the control series which was distinctly convex downwards (Fig.2). The model fitted the hormone data well (Annex Fig.3). The convexity is a consequence of the fall in perfusate pyruvate concentration thus lowering the flux through intracellular pyruvate. The concentration of intracellular pyruvate falls slightly and less unlabelled pyruvate is transferred to the lactate pool. A model which alleviates this difficulty is Model E (Annex) in which the perfusate pyruvate goes straight to the tricarboxylic acid cycle without mixing with the other pyruvate in the heart ( i . e . R, of Model D points to tricarboxylic acid). Although there are serious conceptual difficulties about this model it gives a very good fit to all the tracer data (Annex Fig.4).

Any model with general validity ought to describe the heart metabolism throughout the per-

Fig.2. A d o g wmputer simulation using the results from control hearts, of a four-compartment system for lactatelpyruvate exchange in heart (Model D in text). The open symbols represent experimental values 5 S.E.M. for lactate (o), perfusate pyruvate (0) and heart pyruvate (0). The symbols

(0) represent the values given by the model

fusion and not just in the 30-60min interval. Model D applied to chemical data collected over 2 h of perfusion [l] reproduces the true courses of lactate and pyruvate with remarkable fidelity except that the pyruvate peak is reached a little too early (Annex Fig.5). Since we have no evidence to justify the assumption that the decline in R, should start a t zero time we tried a series of simulations in which the decline was delayed for 10, 20 or 30 min. Model D now behaves in a satisfactory manner (Annex Fig.6). Moreover the period of delay before R, begins to decline appears to be 20min in the absence, and 10min in the presence, of pyruvate added at zero time. This again suggests that pyruvate itself contributes to the decline of R,. That the decline is not apparent from zero time argues against the influence of an added inhibitor such as parapyruvate [4].

Model E on the other hand did not behave well over the whole perfusion period being unable to reproduce the constancy of the lactate pool (Annex Fig. 7). To confirm the essential correctness of Model D and eliminate Model E, an experimental test of a predicted difference between them was carried out. In control hearts R, is negligibly small and so Model E predicts that label added to perfusate pyruvate would not appear in lactate while Model D permits this. Although in rat hearts perfused with pyruvate, more lactate is formed than can be accounted for by the catabolism of glucose, this indirect evidence may only apply to high extracellular pyruvate concentrations [17,18]. When such an experiment is done, with the addition 0.1 pmol x ml-1 x g-1 [2J*C]pyruvate at zero time,


14C is found to accumulate in lactate at a rate con- sistent with the transfer ratea for model D set out below (Fig.3).

100

50

- N 0 u c ._ cn .- L m

Q P

- 10 5

-I

5

looor \ - 600 -

I - .- - 0 E 3. ..-

Y)

-. 400 - .- U Y - .- 3. + ._

2 100 - ” L .- - u a, Q

._

60 - rn

40 -

Transfer Rates in the Pyruvate-Lactate Pool System

The values of the rate parameters and constants obtained [13] using Model D are given in Table 3. These will need slight adjustment when the relatively small alanine-pyruvate exchange is taken into con- sideration (unpublished work). In all models the true size of the highly-labelled intracellular pyruvate pool is not directly ascertainable and the computed value varies with the rate constants relating to it. For this reason and also because “concentrations” in cell pools are quoted per g wet tissue it seems sensible to also give values for the rates of transfer between compartments (Table 3).

A

I I I I 0 10 20 DISCUSSION

The attempts to analyse tracer kinetics after accurate measurement of specific activity in several compartments a t a small number of time points has been gratifyingly successful. Accurate chemical flux measurements are also necessary and together with suitable computing methods [13] a satisfactory analysis of quite a complicated system can be made. Moreover the method tests quantitatively, in the

Perfusion t ime ( m i n )

Fig.3. Intewity of labelling in perfusate pyruvate (0): perfusate lactate ( A ) and perfusate CO, (0) after introdu.ctzon of 0.1 rnM [2-14C]pyruvate (specific activity 100 dis. xs-lx pmol-l) at zero perfusion time. The solid lines represent the predicted labelling patterns according to Model D in the text, with parameter values taken from Table 3. The broken line represents the predicted labelling in perfusate pyruvate according to Model E. The symbols (0 ) represent the values

given by the model

Table 3. Parameters derived directly front the model beat fitting the tracer and chemical fluxes Values aasooiated with the changing perfusate pyruvate pool have been quoted at 3 times where appropriate: at 30 minl 60 mina and zero times. Note that the rate constants for intracellular pools refer to metabolite “concentrations” quoted per

.g tissue not per ml cell water. Reaction numbers 1,2 etc. refer to the reactions in Model D with rates R,, R, etc.

Rate constants for Transfer rates for Reaction in Model D

Control Insulin Growth hormone Control Insulin Qrowth hormone

min-l @ma1 . min-‘ * g-I

2.12 2.55 1.92 1.50 (100) 0.97 0.262

56.63

18.68 0.055

1 2 3 48 5

170.7

154.3 0.024

114.2

146.95 0.021

2.17 1.77

1.10 0.77 1.42 1.15 (91) 0.60 0.302

- 0.03l 0.015a 0.66l 0.332

0.715 0.52 (41) 0.36l 0.042

0.01181 0.05 0.073 0.0

0.00931 0.03 0.041 0.11

0.0161 0.03

1 (decay constant) 6

0.025 0.0046 0.0 t.221

0.41 0.0 0.051 0.0 0.097 7

Irmol/g

Labelled pyruvate 0.039 0.0125 0.010 pool in heart

Vnlabelled pool 0.054 (5S0/,,) 0.0365 (74O/,,) 0.024 (71°/,,)

In triose units with percentage of total glucose flux in parentheses.

Vo1.36, No.Z.1973 J. MOW~RAY and J. H. OTTAWAY 375

Table 4. Cmputed rate8 of conversion of lactate to p v d e The values are quoted in pmol x min-1 x g wet tissue-1. Calculation of lactate dehydrogenaae activity for lactate oxidation was based on the method of Schwert and Winer [19] with

Vr [Lac] [NADI 3.8 x 10“ + 1W [Lac] + 0.17 [NAD] + [Lac] “AD] V =

at pH 7.05 and 28.5 “C. V f for 1 mole catalytic site is 2000 mol x min-l x 1-I. Maximum lactate dehydrogenase activity in rat heart [20,21] corrected for pyruvate inhibition [22] is 1115 pmol xmin-lxg-l. V for 1 mole catalytic site at pH 6.5 is 16000 mol x min-1 x 1-1 [19] so that the fraction of a mole of catalytic site present is (1.115/16000) or 7 x 10-5 in 1 g heart. This will give v in mmol x min-l x ml heart water-1. This fraction agrees quite well with that calculated from the data of Fellenberger et al. [23] for 0.25 mM pyruvate, pH 7.5 in unknown buffer. A 2-fold increase in rate has been allowed for the temperature difference between

28.5 “C and 37.5 “C

Actual rates Bates corrected to 1 mM lactate in heart water Rate

Control Insulin Growth hormone Control Insulin Growth hormone

pmol x min-l x g-I

Exchange (Rz) 1.73 2.55 0.77 1.36 0.60 0.53 To perfusate at 30 min (R,) 0.36 0.97 0.60 0.28 0.23 0.41

Total 2.10 3.62 1.37 1.64 0.83 0.94

Calculated from lactate dehydrogenaae activity in heart extracts 2.02 5.25 2.02 1.59 1.59 1.69

intact cell, our conception of intraceuular organisation. In this study, a well-researched area of metabolism was deliberately chosen and the results demonstrate clearly our ignorance of the extent to which “soluble systems’’ are compartmented in cardiac cytoplasm a t least.

Comparison of Lactate Dehydrogenuse Flux and Measured Tissue Activity

The rate equation, and constants, quoted by Schwert and Winer [19], in their very full treat- ment of lactate dehydrogenase kinetics may be used. Accepting Klingenberg’s report [24] that somewhat less than half the total (NAD + NADH) of heart is in the cytoplasm, values for the lactate dehydrogenase activity a t the measured extract concentrations [I] can be calculated. These values are set out along with the actual rates in Table 4. The control results agree remarkably well while the hormone results are 68O/, of those calculated from lactate dehydrogenase activity. When the data, are normalis- ed to 1 mM lactate in heart water, the hormone exchange rates are very similar. If the differences in the rate of rise and fall in pyruvate level [i] are taken into account it is probable that the “initial” value for the rate of oxidation of lactate to perfusate pyruvate was about the same in all three series of experiments viz. 0.4-0.5 pmol x min-l x g-l. Thus only the lactate dehydrogenase activity in the exchange compartment appears to have been reduced by adding hormones which suggests that this moiety of heart lactate dehydrogenase operates in a cyto- logically distinct space. From the data of Winer and

Schwert [25] it may be calculated that the NAD concentration would have to drop to I/40th of its original value to reduce lactate dehydrogenase activity by 500/,. There is no evidence for such a gross change [1, 261. It seems possible, however, that the observed rise in free NADH concentration (see below) was enough to produce an increase in “dead-end” complex lactate dehydrogenase - NADH - lactate. It is interesting that the finite rate of exchange of isotope between lactate and pyruvate implies a significant concentration of central com- plexes (cf. [27]).

The Activity of Pyruvate Dehydrogenase Rather little work has been done on the transport

of pyruvate into mitochondria [28,29] ; the evidence so far suggests that it proceeds by simple diffusion [30,31]. It follows, therefore, that the constant R, which increases dramatically in the presence of both insulin and growth hormone (Table 3) probably reflects in some way, the activity of pyruvate dehydrogenase. Strong independent evidence for an increase in activity of the complex comes from the fact that the rate of pyruvate oxidation remains high after perfusion with hormones while the overall p p v a t e concentration in the heart falls by half (insulin) or two-thirds (growth hormone).

Although there are no reports of inaulin activat- ing pyruvate dehydrogenase in heart or skeletal muscle, cf. [32], such an activation has been reported for the adipose tissue enzyme [29,33,34]. There is good evidence to show that the oxidation of fatty acids lowers pyruvate oxidation in muscle [15,


35-37]. Acetyl-CoA, known to be a powerful inhibitor of pyruvate dehydrogenase in VitTO [38,39] is considered to be the effective inhibitor [40]. By contrast our hearts were oxidising pyruvate almost exclusively during the tracer experiment [l]. This high proportion of the energy requirement coming from pyruvate is not in accordance with the experience of others [16] particularly for hearts from fasted rats but we are able to confirm it by direct measurement of the specific activities of tricarboxylic acid cycle intermediates and CO, ([S]; unpublished results). High rates of pyruvate oxidation are not associated with high acetyl-CoA concentrations in heart. In hearts from fed rats [41,42] or from fasted rats perfused with insulin [41], the concentration of acetyl-CoA is too low to be measurable. If acetyl-CoA/CoA ratio is inversely related to mitochondria1 NAD/NADH ratio (see below) as it is in liver [43], then acetyl-CoA should be higher in the presence of insulin than in its absence. NADH also known as an inhibitor of pyruvate dehydrogenase [44] increases (see below) in the presence of the hormones. It thus seems that the observed pyruvate dehydrogenase activation cannot be due to removal of product inhibition, The level of free CoA in heart is rather high [33].

It has been shown that active (dephosphorylated) pymvate dehydrogenase comprises about one sixth of total complex in hearts from fasted rats [3(i]. If in our experiments reactivation was complete, the agreement with the 7- to Sfold increase in R, which we observed would be rather good. Randle and his coworkers [29] favour an indirect mediation of hormone action on pyruvate dehydrogenase by 3’:5’-AMP, but insulin only has a marginal effect on 3’:5‘-AMP levels in muscle [32]; while growth hormone, which appears to be as effective as insulin in this respect, is thought to act through the synthe- sis of enzymes concerned with 3‘:B‘-AMP metabolism [45,46], rather than by immediate effects on the nucleotide concentration.

The Decline of Influx to Perfusate Pyruvate It is pertinent to ask whether it is the activity

of lactate dehydrogenase or a leak of pyruvate that declines during the perfusion. The model used implies the former, but a declining pyruvate leak would be compatible with the model provided the pyruvate lost did not mix with either of the other pyruvate pools after its formation from lactate. The only direct evidence, suppression of pyruvate efflux by fatty acid, is inconclusive. A more theoretical approach was tried: the rate of lactate oxidation was kept constant (R, + R, e-It = constant) so that more newly formed pyruvate was transferred to the intracellular pool as the efflux to perfusate decreased. This model fitted the bulk

of the data quite well, but the flux of pyruvate to the tricarboxylic acid cycle and consequently the influx from glucose were forced to levels much higher (e.g. 4 times) than experimental values. Moreover perfusate lactate varied excessively and so it is tentatively concluded that the decline in R, represents a decrease in heart lactate dehydrogenase activity.

The Relationship of Perfusate Pyruvate to Heart Metabolism

No model in which perfusate pyruvate came from an intracellular pyruvate pool was in accord with the tracer data and it appears that pyruvate flux is unidirectionally into the tissue when it occups. Confirmation that pyruvate from phosphoewl- pyruvate does not leave the heart while that produced from lactate oxidation can reach extracellular fluid comes from observations of Henderson et al. [47] that there was negligible pyruvate efflux from hearts perfused with glucose whereas hearts perfused with lactate alone produced 0.23 pmol/min pyruvate, very like our value in the first few minutes of perfusion [l]. This conclusion from the tracer data that pyruvate flux is unidirectional fits well with the observations that the ratio of intracellular to extracellular pyruvate can vary from less than unity [1, 12,181 to greater than unity [l] and would support the probable existence of a pyruvate permease [as]. This has to be set beside the striking conclusion that in the presence of insulin all pyruvate entering the heart is reduced to lactate; in the absence of insulin none is reduced to lactate (i.e. only via the intracellular pyruvate pool). While this could be explained by invoking a special insulin-activated permease, it might as easily be the result of the availability of NADH in the neighbourhood of this cLexternaUy oriented” lactate dehydrogenase. In the absence of insulin this reduced NAD is presumably immediately reoxidised by another redox couple which would fit with the observation that insulin increases not only the absolute amount of glucose transformed to lactate but also the fraction transformed. Further, insulin has no effect on lactate metabolism by heart in the absence of glucose but converts a net uptake to a considerable net output in the presence of glucose [49].

The Redox Potential in Perfused Rat Heart The modelling of the tracer results have clearly

suggested the reason for the observation [1,12,47] that extracellular [lactate]/[pyruvate] ratio cannot directly reflect cardiac intracellular redox potential : this is that neither of the pyruvate pools in the heart lose any material to the perfusate. Our results show that the extracellular pyruvate is a function

Vol.36, No.2,1973 J. MOWBRAY and J. H. OTTAWAY 317

of extracellular lactate which is itself a resultant of lactate brought to heart from other tissues.

The rate constants R, & R, (dimensions, time-l [13]) can be used to evaluate [NAD]/[NADH] and [lactate]/[pyruvate] ratios in the exchange compartment if they are first converted to pseudo-first- order chemical rate constants (dimensions, vol./time). All concentrations are expressed per g weight of heart which avoids the difficulty of the actual concentrations in cell water. Thus R values associated with tissue pools are multiplied by 1 and those associated with perfusate pools are multiplied by 48.42 (average volume of perfusate) to yield the chemical rate constants.

The calculation obviously depends on the validity of the model. It is worth noticing that values of the constants depend very largely on measurements of chemical flux and radioactivity in the perfusate since the estimation of radioactivity in the heart pyruvate carried only one-fifteenth of the total weight in the computations. It has been assumed that since lactate dehydrogenase catalyses a true equilibrium and since lactate-pyruvate exchange in the heart was in equilibrium as shown by the constancy of perfusate [lactate], then :

P, [NAD] [lactate] = P, [NADH ][H+] [pyruvate].

Now the apparent Grst-order rate constants (PI‘ and P2’) estimated are P,’ = P, [NADH] [H+] and

P,’ - [NAD]. Taking the value of K as 5.3 x l O - l 2 M [50] and [H+] as lO-’M the ratios calculated in the presence and absence of hormones are given in Table 5. The [NAD]/[NADH] ratio in the control group is of the same order as that quoted for heart [12] and for liver [5] from apparent [lactate]/ [pyruvate] ratios, but the reduction of the ratio by insulin is far greater than reported by Opie and Mansford for non-working hearts [12]. Growth hormone has reduced the ratio much like insulin.

Given these values, the free [lactate]/[pyruvate] ratio in the exchange compartment can be calculated. P,‘ and Pi, while not independent of chemical estimation of lactate and pyruvate in perfusate, are heavily dependent on tracer fluxes. Heart lactate pool size is purely a chemical estimation and so the good agreement between observed and calculated ratios (Table 5) was not a foregone conclusion. The discrepancy in the growth hormone case is probably due to a slight underestimation of R, which some modelling studies suggest is, in turn, possibly due to the assumption that all lactate in the heart mixes essentially instantaneously. The discrepancy is not serious.

Given the overall [NAD]/[NADH] ratios [l] the mitochondria1 ratios may be calculated on the assumption that the exchange compartment value

P,’ = P, [NAD]. Thus K = P,’ * [NADH] [H+]/

Table 5. NADINADH ratios and lactatelpyruvate ratios computed from rate constants

These values are ratios of unbound reactants and products, using the value 5 . 3 ~ 1 0 - ~ for the equilibrium constant a t pH 7 of the lactate dehydrogenase reaction. For the method

of computation, see text

Ratio Growth Control Insulin hormone ~~

NADINADH ratio in exchange compartment computed from P: and P i 877 130 169

Lactate/pyruvate ratio in exchange compartment computed from P i and Pz’ 21.3 145 111.5

Ratio of lactatelg heart to labelled pyruvatelg heart 19.1 141 76

NAD/NADH ratio in mitochondria, based on as- sumptions in text 20.5 6.0 6.4

applies to the whole cytoplasm (cf. [12]) and that total cytoplasmic nucleotide is 40°/, of tissue amounts [24]. The value in the presence of hormones is very close to that found in liver [51].

Cytoplasmic Comprtmentation in Rat Heart In all three series of experiments there has been

unequivocal evidence for an unlabelled pool of pyruvate. Since lactate exists as a single pool and is accepted to be ubiquitous [47] the implication is that lactate dehydrogenase must be localised. Sup- port for this idea comes from the use of gel-based staining techniques which show that lactate dehydrogenase and several other enzymes are concentrat- ed in the I-bands of flight and skeletal muscle [52-551. The same pattern has been shown to be true for rat heart [56].

Further since perfusate pyruvate does not have the characteristics necessary for the intracellular pyruvate formed from lactate we have been forced to divide the lactate dehydrogenase into “externally oriented” and “exchange” compartments. This division may be more apparent than real since a pyruvate transport phenomenon may be involved (see above). Significantly the transverse invaginations of sarcolemma called T-tubules run between the myofibrils at the level of the Z-discs in cardiac muscle [57] and thus would provide easy immediate access to the extracellular space to pyruvate formed from lactate in the I-band.

The existence of at least two intracellular pyruvate pools implies also that pyruvate b a s e must be localised. h o l d et al. [58] have reported that F-actin contains two binding sites for pyruvate kinase. In view of the fact that hormones induced

378 Effects of Hormones on Pyruvate Flux in Heart Eur. J. Bfochem.

the influx of dabelled pyruvate into the labelled pool, it is an attractive possibility that these asgents altered pyruvate kinase binding. Attempts are being made to test this experimentally (D. R. Jarvie, unpublished work).

Although this study refers to cardiac muscle, the evidence that many glycoIytic enzymes are localised [59] fosters a strong suspicion that the dual organisation of lactate and pyruvate metabolism may extent back up the Embden-Myerhof pathway as has been suggested for rat diaphragm muscle C601.

The Effects of Insulin and Growth Hormone Although growth hormone has been recognised

to have “insulin-like” actions [61-631, it was a surprise to find the hormones so alike. Both apparently activate pyruvate dehydrogenase, par- tially inactivate lactate dehydrogenase, route pyruvate efflux from glucose through the intracellular labelled pool, lower the cytoplasmic and mito- chondrial redox potentid and increase the glycogen level. Only the facilitation of perfusate pyruvate reduction to lactate, the increase in glucose uptake and the higher steady-state perfusate lactate concentrations were characteristic of insulin and not growth hormone. Growth hormone alone increases the rate constant for pyruvate uptake from perfusate without reducing thia pyruvate obligatorily to lactate.

ANNEXES The following documents have been deposited at the

Archives Originales du Centre de Documentation du C.N.R.S (26 Rue Boyer, F-75020, Paris, France) where they may be ordered as microfiche or photocopies. Reference No.

Annex, Fig. 1. Decrease in concentration of pyruvate in the perfusate during the period 30-60 min of perfusion.

Annex, Fig. 2. Attempted fit of Model C to experimental data from (A) control and (B) insulin hearts.

Annex, Fig.3. Fit of Model D to experimental data from (A) insulin and (B) growth hormone hearts.

Annex, Fig.4. Fit of Model E to experimental data from control hearts.

Annex, Fig.5. Digital computer simulation of the be- haviour of perfusate lactate and pyruvate over 120 inin as prediczed by Model D.

Annex, Fig. 6. Digital computer simulation of the beha- viour of perfusate lactate and pyruvate over 120rnin as predicted by Model D when pyruvate was (A) absent and (B) present at zero time. The decay of R, was allowed to start at 10,20 and 30 min.

Annex, Fig. 7. Digital computer simulrttion analogous to that in Annex Fig.6, using Model E.

Annex, Model C. Four-compartment system in which perfusate pyruvate exchanges only with labelled intracellular pyruvate.

Annex, Model E. Four-compartment system in which perfusate pyruvate exchanges with lactate and can efflux directly to the tricarboxylic acid cycle without entering the labelled tissue pyruvate pool.

A.O.-524.

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Documents

The Effect of Insulin and Growth Hormone on the Flux of Tracer from Labelled Lactate in Perfused Rat Heart