Studies on the Effects of Coenzyme A-SH: Acetyl Coenzyme A, Nicotinamide Adenine Dinucleotide: Reduced Nicotinamide Adenine Dinucleotide, and Adenosine Diphosphate:Adenosine Triphosphate Ratios on the Interconversion of Active and Inactive Pyruvate Dehydrogenase in Isolated Rat Heart Mitochondria

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DLCarnitine and acetyl-DLcarnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP

The content of coenzyme A-SH (CoASH) and acetyl-CoA of suspensions of rat heart mitochondria was stabilized by the addition of DLCarnitine and acetyl-DLcarnitine, in the presence of the respiratory inhibitor rotenone. The mitochondrial content of NAD+ and NADH was similarly stabilized by the addition of acetoacetate and DL-3-hydroxybutyrate, and the content of ADP and ATP was imposed by the addition of these nucleotides to the mitochondrial suspension, in the presence of uncoupling agent and oligomycin, to inhibit ATPase. Under these conditions, mitochondrial CoASH/acetyl-CoA, NAD+/ NADH, and ADP/ATP ratios could be varied independently, and the effect on the interconversion of active and inactive pyruvate dehydrogenase could be studied. Decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios were shown to be inhibitory to the steady state activity of pyruvate dehydrogenase, and this effect is described at three different ADP/ATP ratios and different concentrations of added MgCl,. A new steady state level of activity was achieved within 10 min of a change in either CoASH/acetyl-CoA or NAD+/NADH ratio; the rate of inactivation was much higher than the rate of reactivation under these conditions. Effects of CoASH/acetyl-CoA and NAD+/NADH may be additive but are still quantitatively lesser than the changes in activity of pyruvate dehydrogenase induced by changes in ADP/ATP ratio. The variation in activity of pyruvate dehydrogenase with ADP/ATP ratio is described in the absence of changes in the other two ratios, conditions which were not met in earlier studies which employed the oxidation of different substrates to generate changes in all three ratios.
The mammalian pyruvate dehydrogenase complex has been shown to be subject to regulation by a process of phosphorylation and dephosphorylation (l-3). Phosphorylation, and consequent inactivation, requires ATP, whereas ADP promotes a net dephosphorylation of the enzyme by inhibiting the activity of the pyruvate dehydrogenase kinase (4) in the presence of an active phosphatase. Since these findings much attention has been devoted to an investigation of the dependence of pyruvate dehydrogenase activity upon the ADP/ATP ratio of the mitochondrial matrix (5-7). Although generally a good correlation has been shown between enzyme activity and ADP/ATP ratio, there have been anomalies (5, 7). An explanation of these may be the recent findings by Pettit et al. (8) and Cooper et al. (9) that pyruvate dehydrogenase kinase is also activated by decreased CoASH/acetyl-CoA and NAD+/NADH ratios. This is of great significance in that it may provide a mechanism for the inhibition of pyruvate oxidation by fatty acids which is seen in the perfused heart (10) and which is associated with decreased whole tissue CoASH/acetyl-CoA and NAD+/NADH ratios. The experiments of Pettit et al. (8) employed a purified pyruvate dehydrogenase preparation, containing kinase, to which an arbitrary amount of phosphatase is added in the cuvette. It seemed important to demonstrate that the pyruvate dehydrogenase complex of rat heart is indeed subject to regulation by CoASH/acetyl-CoA and NAD+/NADH ratios when present in the mitochondrion, in the presence of endogenous phosphatase and of mitochondrial matrix concentrations of effector nucleotides. Such a study has recently been reported for liver mitochondria by Batenberg and Olson (11) and achieved a partial separation of the effects of the CoASH/acetyl-CoA and NAD+/NADH ratios. Complete separation was not possible as respiring mitochondria were used, and substrate additions tended to change both ratios, in addition to possibly changing ADP/ATP.
The study being presented here aims to demonstrate regulation of pyruvate dehydrogenase in rat heart mitochondria by CoASH/acetyl-CoA, NAD+/NADH, and ADP/ATP ratios and to discriminate between these effecters by using nonrespiring mitochondria, with each of these ratios 5483 enforced by a suitable added "buffer." In this way each ratio can be manipulated at will, independently of the others.

EXPERIMENTAL PROCEDURE
Preparation ofMitochondria-Mitochondria were prepared from the hearts of 6-month male Wistar-derived rats from the Gerontology Research Center aging colony. The method was substantially that described by Chappell and Hansford (12)  suspensions, in which the ratio of added acetoacetate/oL&hydroxybutyrate was varied, and mitochondrial NAD+ and NADH were measured. The results testify to a near-equilibrium at the 3-hydroxybutyrate dehydrogenase reaction and show that the acetoacetate/3-hydroxybutyrate couple can be used to impose mitochondrial ratios of NAD+/NADH in the range of interest. It was predicted that the NAD+/NADH ratios would not be affected by the difference in DL-carnitine/acetyl-DL-carnitine ratio in the two experiments represented in Table II, and this was the case.
Mitochondrial ADP and ATP were not measured, but there is a good reason to believe that in the presence of oligomycin and the uncoupling agent FCCP, the matrix ADP/ATP ratio approximates that in the incubation medium. This is because  (5-7). However, the ADP/ATP ratio has in general been manipulated by using combinations of different oxidizable substrates and inhibitors which may plausibly change CoASH/ acetyl-CoA and NAD+/NADH ratios at the same time (see under "Discussion").
Because of this, this relationship has been reinvestigated, with CoA/acetyl-CoA and NAD+/NADH ratios stabilized. Fig. 3 shows that when heart mitochondria are incubated in the presence of EDTA, pyruvate dehydrogenase activity is exceedingly sensitive to inhibition by ATP. Thus, when ADP/ATP = 1, activity is only 10% of maximal, despite the fact that mitochondrial CoASH/acetyl-CoA and NAD+/NADH ratios are approximately 4.8 and 11, respectively, and not inhibitory. Activities are considerably increased by the presence of 20 mM isobutyrate, which has been shown to inhibit pyruvate dehydrogenase kinase (3) presumably by the same mechanism as pyruvate (4). The picture which emerges is quite different when the incubation medium containing EDTA is replaced by one containing 4.3 mM MgCl, (Fig. 4). The response of pyruvate dehydrogenase activity is now shifted to ADP/ATP ratios close to those found in respiring mitochondria (18,19). Isobutyrate was included in a portion of this study (Fig. 4) and the previous one (Fig. 3) in a realization that pyruvate is present in uiuo and is an effector of pyruvate dehydrogenase kinase (4) Tables I and II, and pyruvate dehydrogenase activity was estimated after being allowed 10 min to achieve a steady state (see Figs. 1, 2). An ADP/ATP ratio of 3.5 was chosen as being a plausible matrix ratio during state 3 (plus ADP) substrate oxidation by isolated mitochondria.
The mitochondrial ADP/ATP ratio in the intact heart is not available. It is seen that changes in either CoASH/acetyl-CoA or NAD+/NADH ratio alone can result in more than 2-fold changes in enzyme activity, and that simultaneous changes in both ratios can result in a 4-fold change. This in viuo, and so this study ( Fig. 7) was carried out in the presence of 0.5 mM dichloroacetate. This is a high concentration compared to that previously found effective in stimulating heart mitochondrial pyruvate dehydrogenase (20), but no concentration gradient will exist across the mitochondrial membrane in the present studies, owing to the presence of an uncoupling agent. It is seen (Fig. 7 and m-3-hydroxybutyrate were added to a total of 50 rmol and in one of the ratios given in Table II. m-Carnitine and acetyl-m-carnitine were added to a total of 10 pmol and in one of the ratios given in Table I 7. The dependence of pyruvate dehydrogenase activity on CoASH/acetyl-CoA and NAD+/NADH ratios, in the presence of 4.3 mM MgCI,, an ADP/ATP ratio of 0.36, and 0.5 mM dichloroacetate. The protocol was exactly as described for Fig. 5 with the exception that the mitochondrial suspension was of 27 mg of protein/ml, and additions of ADP and ATP were of 2.5 and 5 @mol. respectively. The ratio of ADP/ATP found on sampling the incubations was 0:363 + 0.002, and the maximal activity of this preparation was 87 nmol/min/ mg of protein.

This study reports the effect of mitochondrial
CoASH/acetyl-CoA, NAD+/NADH, and ADP/ATP ratios upon the steady state activity of the mitochondrial pyruvate dehydrogenase complex. It does not distinguish between effects on the pyruvate dehydrogenase kinase and the phosphatase. However, it is hoped that the results can be applied to the problem of the effect of lipid upon pyruvate oxidation in the heart. The maximal activity of pyruvate dehydrogenase reported here is similar to that reported by Kerbey et al. (7) for rat heart, although Kerbey et al. employed a slightly higher temperature (30"). It is substantially less than that reported by Chiang and Sacktor (21) for rabbit heart mitochondria, though the difference may be small when the temperature difference is allowed for. The conditions described under "Experimental Procedure" for generating maximal activity of pyruvate dehydrogenase yielded the same results as conditions giving a maximal rate of pyruvate oxidation by respiring mitochondria, i.e. 2.5 mM pyruvate, 1 mM malate, 5 mM phosphate, and 0.5 pM FCCP (not shown). Under these conditions, the rate of 0, consumption at 25" was 0.34 c(g atoms/min/mg of protein, which demands as a minimum a pyruvate dehydrogenase activity of 113 nmol/min/mg if all of the 2-oxoglutarate which is formed leaves the mitochondrion, or 68 nmol/min/mg, if none of it does. A tentative conclusion is that the recovery of pyruvate dehydrogenase from the mitochondria is reasonable. The rates of inactivation and reactivation of pyruvate dehydrogenase reported here (Figs. 1 and 2) are very much faster than those found in rabbit heart mitochondria (21). A possible reason for the more rapid reactivation in the present study is that incubation media contained high [K+ 1, which is necessary for inhibition of the kinase by ADP (22) and for the effects upon the kinase of CoASH/acetyl-CoA and NAD+/NADH ratios (8). The rapidity of inactivation and reactivation shown in the present study casts doubt upon the adequacy of centrifugation (see e.g., Ref. 7) as a technique for ending mitochondrial incubations prior to pyruvate dehydrogenase estimation.
The present results demonstrate that the effects of CoASH/ acetyl-CoA and NAD+/NADH ratios on pyruvate dehydrogenase interconversion which were shown by Pettit et al. (8) at the level of the purified enzyme also apply to the enzyme in its mitochondrial milieu. Previously this information was not available for heart mitochondria.
Thus, in the very recent study by Kerbey et al. (7) there are anomalies in the correlation between pyruvate dehydrogenase activity and the inverse of the mitochondrial ATP content, and these are cited as evidence for a modulation of pyruvate dehydrogenase interconversion by CoASH/acetyl-CoA and NAD+/NADH ratios. This is plausible, but not clear, and the different effects of CoASH/ acetyl-CoA and NAD+/NADH ratios are not segregated. Thus, addition of octanoate to respiring mitochondria caused greater inactivation of pyruvate dehydrogenase than expected on the grounds of mitochondrial ATP content, but caused large decreases in both CoASH/acetyl-CoA and NAD+/NADH ratios. In liver the picture is clearer, in that similar anomalies in the correlation of pyruvate dehydrogenase activity with the mitochondrial ADP/ATP ratio were originally noted by Taylor et al. (5), and a partial resolution of the effects of NAD+/ NADH and CoASH/acetyl-CoA ratios was subsequently made by Batenburg and Olson (11). Very recently, the latter authors (23) have reported experiments using mixtures of 3-hydroxybutyrate and acetoacetate and mixtures of octanoate and carnitine. Though these experiments establish an effect of CoASH/ acetyl-CoA and NAD+/NADH ratios on pyruvate dehydrogenase interconversion in rat liver, they are quantitatively equivocal, as the mitochondria will oxidize the 3-hydroxybutyrate in the absence of rotenone and will progressively acetylate the carnitine in the presence of octanoate. Thus CoASH/acetyl-CoA and NAD+/NADH ratios will change during the course of the experiment and, judged from Figs. 1 and 2 of the present paper, alterations in pyruvate dehydrogenase activity will lag behind these changes.
The present study also documents the effect of the ADP/ ATP ratio upon pyruvate dehydrogenase activity. Previously published experiments have altered ADP/ATP at the same time as NAD+/NADH and CoASH/acetyl-CoA ratios, by varying respiratory substrates (5-7). An exception is the study of rabbit heart mitochondria by Chiang and Sacktor (21) where adenine nucleotides were added in the absence of substrate. However, the object of that study differed from that of the present one, in that it dealt with rates of inactivation or reactivation, and NaF was added to inhibit phosphatase. The present study deals with steady state levels of active pyruvate dehydrogenase, a resultant of the balance of kinase and phosphatase. The dependency on ADP/ATP which is obtained (Figs. 3 and 4) is markedly affected by the presence of MgCl,, though only in the range 0 to 4.3 mM added MgCl, and not above that (results not shown). Free [Mg*+] will be considerably less than 4.3 mM, as there is 3.8 mM total adenine nucleotide present in these studies. Arguments based on the aconitase equilibrium and on the binding of Mg*+ to known ligands have suggested about 1 mM free MgZ+ in a number of tissues (24), and so these experimental conditions were thought reasonable. The dependency of pyruvate dehydrogenase activity upon ADP/ATP ratio was also affected by the presence of the carboxylates isobutyrate and dichloroacetate. Pyruvate could not be added in these studies, being incompatible with the buffering of NAD+/NADH and CoASH/acetyl-CoA ratios. However, studies are in progress which utilize pyruvate as respiratory substrate and attempt to correlate the inactivation of pyruvate dehydrogenase seen on adding palmitoylcarnitine, with the changes which occur in the modulator ratios discussed in this paper. for expert technical assistance and Dr. Bertram Sacktor for his interest in this work.