Regulation of Pyruvate Dehydrogenase in Muscle INHIBITION BY

SUMMARY The active and inactive forms of pyruvate dehydrogenase were assayed in a mitochondrial extract prepared from rat skeletal muscle. The active form of this enzyme was increased almost 3-fold upon preincubation with 10 mM Mg++ and Ca++. There was a decrease of approximately 30% when 1 mM ATP and 0.5 mu Mg++ were present in the original preincubation mixture. If citrate (5.0 mM) was added to the preincubation mixture containing 10 mu Mg+f and Cat+, the activation of pyruvate dehydrogenase was almost completely prevented. The concentration of citrate required to reduce the rate of activation of pyruvate dehydrogenase by 50% was approximately 2.0 mM in this system. Citrate did not inhibit the assay system for activated pyruvate dehydrogenase. Once the activation of pyruvate dehydrogenase was complete, addition of 5 mu citrate to the preincubation mixture did not decrease this activity. Increasing the concentrations of Mg++ and Ca++ from 10 to 20 mu did not overcome the citrate (5 mnrr)-induced prevention of pyruvate dehydrogenase activation.

From the Department of Medicine, University of Toronto School of Medicine, Toronto, Ontario, Canada M&S lA8 SUMMARY The active and inactive forms of pyruvate dehydrogenase were assayed in a mitochondrial extract prepared from rat skeletal muscle.
The active form of this enzyme was increased almost 3-fold upon preincubation with 10 mM Mg++ and Ca++.
There was a decrease of approximately 30% when 1 mM ATP and 0.5 mu Mg++ were present in the original preincubation mixture. If citrate (5.0 mM) was added to the preincubation mixture containing 10 mu Mg+f and Cat+, the activation of pyruvate dehydrogenase was almost completely prevented. The concentration of citrate required to reduce the rate of activation of pyruvate dehydrogenase by 50% was approximately 2.0 mM in this system.
Citrate did not inhibit the assay system for activated pyruvate dehydrogenase. Once the activation of pyruvate dehydrogenase was complete, addition of 5 mu citrate to the preincubation mixture did not decrease this activity.
Increasing the concentrations of Mg++ and Ca++ from 10 to 20 mu did not overcome the citrate (5 mnrr)-induced prevention of pyruvate dehydrogenase activation.
It is concluded that citrate can prevent the activation of pyruvate dehydrogenase by a mechanism independent of Mg++ and Ca++ chelation. Some possible physiological implications are discussed.
It is now well established that the activity of pyruvate dehydrogenase (EC 1.2.4.1) in mammalian liver, kidney, cardiac muscle, adipose tissue, and brain is regulated by a phosphorylationdephosphorylation cycle (l-6). Phosphorylation (and inac- Foundation. However, this mechanism has not )-et been confirmed for the regulation of skeletal muscle pgruvate dehydrogenase. It now appears that such an interconversion betw-een the inactive and active forms of pyruvate dehydrogenase plays a crucial role in the regulation of the activity of this enzyme under a number of different hormonal and dietary st,ates (4,5,(9)(10)(11). The detailed mechanism whereby these altered ratios of active to inactive forms of pyruvate dehydrogenase can be achieved under these different metabolic states, however, has yet to be elucidated.
Dietary states which favor the interconversion of pyruvate dehydrogenase to the inactive form in liver: such as starvation or feeding a high fat diet (9)) also cause a depression of pyruvate oxidation in cardiac and diaphragm muscle (12). This depression of pyruvate oxidation in muscle is associat,ed with elevated tissue citrate levels (12-14).
Citrate (1.0 IllM) has been reported to have no effect on the activation of pyruvate dehydrogenase (7). Higher concentrations of citrate inhibited pyruvate dehydrogenase in rat liver mitochondria (15). The studies conducted by those authors (15), however, could not determine whether citrate inhibited the active form of the enzyme or the interconversion of inactive to active pyruvate dehydrogenase.
Alt.hough this action of citrate did not appear to involve Mg++ chelation, it could have been a result of the chelation of Ca++.
In view of the possible physiological importance of the regulation of pyruvate dehydrogenase by citra.te, the effects of citrate on the activation of pyruvate dehydrogenase in skeletal muscle will be reported.

MATERIALS AXD METHODS
Rats-Skeletal muscle was rapidly disscctcd from the hind limbs of male Wistar rats (110 to 140 g) which were obtained from High Oak Ranch, Goodwood, Ontario.
In all experiments the rats were allowed free access to Purina Lab Chow diet until the time of death (10:00 a.m. Dehydrogenase-In assays of pyruvate dehydrogenase activity, the mitochondria from muscle obtained from 8 to 12 rats were homogenized in ice cold buffer (10 rnM potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol, 1% bovine serum albumin-fat,@ acid free, $1 7.4), as described by Taylor et al. (ll), to give a final mitochondrial protein concentration of 1.0 mg per ml. I'yruvate dehydrogenase activity was determined before and after 1)rcincubation of this enzyme comples for the designated times by the method of Taylor et ai. (11).
Acct\.l-d'oA was assayed fluorometrically by the method of Wieland :urd Weiss (18)  This suggests that there was no interconversion between the active and inactive forms of pyruvate dehydrogenase during the 2-min assay. Skeletal muscle pyruvate dehydrcgenase was found to have a K, for pyruvate of 9.1 PM (Fig. 1C). On the basis of these results, pyruvate dehydrogenase assays were routinely performed over a 2-min incubation period, using pyruvatc concentrations of 0.6 to 0.7 mM and mitochondrial protein levels of 0.2 to 0.4 mg per assay. Maximum activation was achieved by preincubation with 10 rnnf Mg++ and 10 mM Ca+f (Table I).

EJSect oj ATP and Divalent
Ions on Pyruvate Dehydrogenase ilctivity-Prior to assay, muscle pyruvate dehydrogenase was prcincuba,tcd for 15 min at room temperature in the presence of 6081 bhe additions shown in Table II. In contrast to pyruvate dehydrogenase studied in other mammalian systems (7), muscle pyruvate dehydrogenase activity increased only slightly after incubation with 10 mM Mg+f or Mn+f.
However, a substantial increase in pyruvate dchydrogenase activity was observed in the presence of 10 mM Mg++ and 10 rnM Ca++. Under these con- TANGLE  ditions pyruvate dehydrogenase activity was increased approximately a-fold. This increase in pyruvate dehydrogenase activity was consistent with the recent finding of Denton et al. (S), who found that pyruvate dehydrogenase phosphate phosphatase from heart, kidney cortex, or adipose tissue was stimulated markedly by the simultaneous addition of Mg++ and Ca++. On the other hand, preincubation of muscle pyruvate dehydrogenase with 1 rnM ATP and 0.5 mM Mg++ (conditions previously shown to stimulate pyruvate dehydrogenase kinase in other tissues) resulted in a small but significant decrease in pyruvate dehydrogenase activity (Table  II). These findings are consonant with the interpretation that muscle pyruvate dehydrogenase activity may be regulated by a phosphorylationdephosphorylation cycle similar to that already established in other mammalian tissues (l-6).  (Table III). When pyruvate dehydrogenase was fully activated by 10 mM Mg++ and Ca++, no effect was observed upon subsequent addition of 5 MM citrate.
This suggested that citrate could not decrease the quantity of the active form of pyruvate dehydrogenase under these conditions, nor did it inhibit the assay for pyruvate dehydrogenase (also see Table V). Citrate added to the preincubation medium resulted in a decrease in acetyl-CoA formation (15 f 1.3 versus 10 f 1.6 nmoles of acetyl-CoA per assay, n = 8, p < .05). It is clear that inhibition of pyruvate dehydrogenase by citrate did not result from elevated acetyl-CoA levels.
It has recently been reported (20) Table  IV.
Increasing the concentrations of these various divalent cations had no appreciable effect on the observed citrate inhibition.
The specificity of the citrate inhibition described was investigated with the use of the various citrate analogues listed in Table IV. These compounds, like citrate, had no direct effect upon the active form of pyruvate dehydrogenase (Table V). Of the tricarboxylic acids tested, only citrate, (-) hydroxycitrate, fluorocitrate, 2-ethylcitrate, and 2-methylcitrate inhibited the activation of pyruvate dehydrogenase by a mechanism independent of metal chelation (Table IV).
In contrast, the inhibitory effects observed in the presence of tricarballylate and 1,2,3-tricarboxpbenzene were abolished by the addition of 20 m&f Ca++. From these experiments, it is evident that the structural requirement for citrate inhibition of pyruvate dehydrogenase requires more than simply a tricarboxylic acid. However, the importance of metal chelation by this group of compounds is underscored by the reversal of tricarballylate-and 1,2,3tricarboxybenzene-induced inhibition of pyruvate dehydrogenase by Mg++ and Ca++.
Pyruvate dehydrogenase activity in liver is decreased during conditions in which long chain fatty acyl-CoA levels and rates of fatty acid oxidation are increased, as during fasting, fat feeding, and diabetes mellitus (9). This probably represents net conversion of pyruvate dehydrogenase to its phosphorylated form. It has recently been demonstrated that elevated long chain fatty acyl-CoA levels inhibit the mitochondrial citrate transporter (21, 22). As a result, mitochondrial citrate levels should rise, thereby promoting inactivation of pyruvate dehydrogenase. The observation that insulin causes pyruvate dehydrogenase activity to increase (4,5,10,11,23) can also be interpreted in light of this observation.
Insulin administration results in a decrease in hepatic (24, 25) and adipose tissue (26, 27) long chain fatty acyl-CoA levels, thereby reducing the inhibition of the 6OS3 mitochondrial citrate transporter (21,22). Citrate concentration in the mitochondria should consequently fall, resulting in activation of pyruvate dehydrogenase, if results in Table III  can be extrapolated to liver or adipose tissue, or both. In this regard, it is of interest to note that inhibitors of the mitochondrial citrate transporter, such as 1,2,3-tricarboxybenzene, have been reported to inhibit pyruvatc oxidation in rat liver mitocl1ondria.i Citrate inhibits the interconversion of the inactive form of pyruvate dehydrogenase to the active form of the enzyme. Whether citrate exerts its inhibitory effect by influencing the phosphorylation-dephosphorylation mechanism is now being investigated.