Relative importance of pyruvate dehydrogenase interconversion and feed-back inhibition in the effect of fatty acids on pyruvate oxidation by rat heart mitochondria

ARCHIVES OF BIOCHEMISTRY Vol. 191, No. 1, November,

AND

Relative Importance Feed-Back Inhibition

of Pyruvate Dehydrogenase Interconversion and in the Effect of Fatty Acids on Pyruvate Oxidation by Rat Heart Mitochondria

RICHARD Laboratory

BIOPHYSICS

pp. 65-81, 1978

G. HANSFORD’

of Molecular Aging, Gerontology Institutes of Health, Baltimore Received December

AND

LINDA

COHEN

Research Center, National Institute City Hospitals, Baltimore, Maryland

on Aging, National 21224

22, 1977; revised June 30, 1978

Rat heart mitochondria have been incubated with concentrations of pyruvate from 50 to 500 FM as substrate in the presence or absence of an optimal concentration of pabnitoylcamitine and with respiration limited by phosphate acceptor. The rate of pyruvate utilisation has been determined and compared with the amount of active (dephosphorylated) pyruvate dehydrogenase measured in samples of mitochondria taken throughout the experiments and assayed under V,., conditions. At a given pyruvate concentration, differences in pyruvate utilization as a proportion of the content of active pyruvate dehydrogenase are attributed to differences in feed-back inhibition and interpreted in terms of the ratios of mitochondrial NAD’/NADH and CoA/acetyl-CoA. Under most conditions, differences in inhibition can be attributed to differences in the CoA/acetyl-CoA ratio. Feed-back inhibition is very pronounced in the presence of pahnitoylcarnitine. These parameters are also examined in the presence of dichloroacetate, used to raise the steady-state levels of active pyruvate dehydrogenase in the absence of changing the pyruvate concentration, and in the presence of various ratios of carnitine/acetylcamitine, which predominantly change the mitochondrial CoA/acetyl-CoA ratio. The latter experiment provides evidence that a decrease in mitochondrial NAD’/NADH ratio from 3.5 to 2.2 essentially balances an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in modulating enzyme interconversion, whereas the change in CoA/acetyl-CoA ratio is preponderant in effecting feed-back inhibition. Increasing the free Ca” concentration of incubation media from lo-’ to lo-’ M using CaCln-ethylene glycol bis(P-aminoethyl ether)-N,h”-tetraacetic acid buffers is shown to increase the steady-state level of active pyruvate dehydrogenase in intact mitochondria, in the absence of added ionophores. Moreover, this activation is reversible. Effects of Ca” ions are dependent upon the poise of the enzyme interconversion system and were only seen in these experiments in the presence of palmitoylcarnitine.

The availability of fatty acids, ketones, or acetate to the rat heart results in a substantial decrease in the rate of oxidation of pyruvate (l-3). Similarly, the addition of long-chain acylcarnitine derivatives to isolated rat heart mitochondria results in an inhibition of pyruvate oxidation (4). These findings appeared to be readily explained on the basis of a feed-back inhibition of pyruvate dehydrogenase by two of the endproducts of the reaction, NADH and acetylCoA, which could be shown in studies with the purified enzyme (5-8). Thus, the conditions outlined above which led to a dimi’ To whom all correspondence

should be addressed.

nution in flux through pyruvate dehydrogenase also led to large increases in acetylCoA content, and decreases in CoA content. However, more recently a second, quite separate, means of pyruvate dehydrogenase control has been demonstrated, involving phosphorylation of the enzyme by a kinase to yield an inactive form and dephosphorylation by a phosphatase, which restores activity (9, 10). The steady state tissue content of active pyruvate dehydrogenase (referred to in this paper as PDH,) then refleets a dynamic balance of kinase and phosphatase activities. Recently, it has been shown that the pyruvate dehydrogenase kinase is activated by decreases in the

65 0003-9861/78/1911-0065$02.00/O Copyright 0 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.

66

HANSFORD

ratios of CoA/acetyl-CoA and NAD+/ NADH (11-13) and it has been established that the perfusion of isolated rat hearts with fatty acids, ketones, or acetate leads to a diminished PDH, content (14). In addition, studies with isolated rat heart mitochondria have shown a sensitivity of pyruvate dehydrogenase interconversion to the ratios of mitochondrial NAD’/NADH and CoA/acetyl-CoA (15, 16) and have attempted to explain the effect of palmitoylcarnitine oxidation on PDI% content in terms of changes in these ratios (16, 17). Similar studies have also been made with liver mitochondria (18). Thus, it is now apparent that the effects of fatty acids and ketones on the metabolism of pyruvate by heart muscle may be mediated by two mechanisms: 1) the diminution in the PDH, content of the tissue, which is modulated in part by changes in the CoA/acetyl-CoA and NAD+/NADH ratios, and 2) direct feed-back inhibition of that part of the pyruvate dehydrogenase present in the active form (PDH,) by the end-products acetyl-CoA and NADH. It seems important to understand the quantitative significance of the two modes of inhibition and this can best be done by comparing the content of PDH, with the flux through the enzyme under a variety of conditions. The present paper describes attempts along these lines. Some similar experiments have been done by Kerbey et al. (13), in which the effect of palmitoylcarnitine and acetylcarnitine on the flux through pyruvate dehydrogenase was measured. Unfortunately, the acylcarnitine derivatives led to no change in PDH, content in that work and so only one of the mechanisms of control seen in the intact heart was reconstructed under the particular conditions of their mitochondrial experiments. In a very recent paper from the same group (16), pyruvate dehydrogenase inactivation due to fatty acid oxidation was demonstrated, but no measurements of flux were made. Hiltunen and Hassinen (19) have also compared flux through pyruvate dehydrogens.c;,! with PDH, content in the intact perfused heart and varied these parameters by KCl-induced arrest. The present study complements their work by looking at changes due to fatty acid oxidation and, by using iso-

AND

COHEN

lated mitochondria, allows the measurement of the CoA and acetyl-Ll.4 < mcentrations present within the same
PROCEDURES

Mitochondria were prepared from the hearts of 6month male Wistar-derived rats from the Gerontology Research Center by the method of Chappell and Hansford (20), but using 2.5 mg of nagarse per heart. Preparations were from one animal for pyruvate dehydrogenase and flux determination and from two when mitochondrial coenzymes were estimated. Incubation of mitochondria was at 25’C and in media described for the appropriate table or figure. 02 uptake was recorded using a Clark-type O2 electrode (Yellow Springs, 0.) in a 2-ml glass incubation vessel (Gilson). Samples (50 al) were removed during the incubation, added to 250 111of ice cold stopping solution, of composition described previously (15, 17) and assayed for PDH. content within 10 min, as described previously (15). The stopping solution is intended to lyse the mitochontia and prevent any further pyruvate dehydrogenase interconversion; the pyruvate dehydrogenase assay conditions are designed to give maximal (V,,) activity. At the termination of the incubation (either 5 or 10 min), a l-ml portion was withdrawn and added to 1.0 ml of 16% (v/v) HCIOI. This extract was neutralized as described previously (15) and used for the determination of pyruvate concentration. A key feature of this work was the approximate stabilization of pyruvate at low concentrations during the course of extended experiments (5 and 10 min). To this end, a ratio of 02 uptake/pyruvate consumption was determined for each experimental condition, initially by trial and error. Addition of pyruvate during the experiments on the basis of the recording of 02 uptake then allowed the approximate stabilization of pyruvate concentration. Additions were made each time 10% of the original 02 content of the medium was depleted (5% when the substrate was 50 PM pyruvate). The final values of 02 uptake/pyruvate consumption obtained are tabulated, and vary with the respiratory state and presence of alternative substrates. Typically, the experimentally determined pyruvate concentration at the end of the experiment was below the nominal concentration, because the time of sampling was not related to the time

PYRUVATE

DEHYDROGENASE

of pyruvate supplementation. However, when comparison is made between experiments in which the pyruvate concentration is nominally the same, it is normally very close. Thus, the results for pyruvate concentration at 10 min obtained in the experiments of Table IV using three different mitochondrial preparations, were 33 & 3, 83 f 1, 217 f 3, 449 If; 4 pM for experiments with pyruvate as sole substrate and 42 + 2, 92 f 2, 219 rt 2, 447 f 1 pM for experiments with pyruvate plus palmitoylcarnitine, and are typical. Without supplementation, pyruvate depletion would have been total at the lower concentrations. This technique was used in previous work (17), which gives some additional detail. Flux through pyruvate dehydrogenase was then estimated from pyruvate consumption, both the amount remaining at the end of the experiment and the amount added during the experiment being known with good accuracy. The method is more cumbersome than methods based on 14C02 release from [I-‘%]pyruvate, but the latter were ruled out as not permitting use of low pyruvate concentrations without excessive substrate depletion over 10 min experiments (as pyruvate supplementation is not possible). Experiments at low pyruvate concentrations were judged more informative on pyruvate dehydrogenase interconversion in previous work (17). When mitochondrial coenzymes were to be measured, 10 ml incubations were performed using Ozsaturated medium. This necessitated prior pilot-scale (2 ml) experiments with air-saturated medium in which rates of 02 uptake were determined accurately, followed by pyruvate supplementation to the large scale incubation on the basis of time elapsed. Pyruvate, ADP, and ATP were determined spectrophotometrically in neutralized HClOa-extracts as described previously (15). NAD+ was determined fluorimetrically in the same extracts (15) and CoA and acetyl-CoA were determined by the kinetic method of Allred and Guy (21), as described previously (15). NADH was determined fluorimetrically in neutralized KOH extracts (15). Nagarse was obtained from Enzyme Development Corp., N.Y. and all other enzymes from Boehringer Mannheim Corp., N.Y. Sodium pyruvate was from Calbiochem and was made up freshly in water, assayed, and diluted before use. Palmitoyl-L-carnitine and CoA were from P-L Biochemicals, Inc. D,L-carnitine hydrochloride was from Calbiochem and acetylD,L-CaITIitine

hydrochloride

was from

Sigma.

RESULTS

Inactivation of pyruvate dehydrogenase by palmitoylcarnitine oxidation in state 4’ ’ Abbreviations used: State 4 and state 3 respiration refer to respiration in the absence and presence of ADP, respectively; Hepes, 4-(2-hydroxyethyl)-l-piperazine-ethanesulfonic acid; EGTA, ethylene glycol bis(P-aminoethyl ether)N,N’-tetraacetic acid.

CONTROL

IN HEART

67

and the measurement of flux through pyruvate dehydrogenase. In a previous study from this laboratory (17)) it was shown that pahnitoylcarnitine oxidation by isolated rat heart mitochondria led to inactivation of pyruvate dehydrogenase (i.e., decrease in PDH, content) provided that the concentration of pyruvate in the incubation was kept low. Table I presents results which confirm this observation and show that in the state of controlled or resting respiration obtained in the absence of phosphate acceptor (state 4 of Chance and Williams (22)) no inactivation is obtained when pyruvate is present at 500 PM. Presumably, this reflects inhibition of the pyruvate dehydrogenase kinase as described by Hucho et al. (23) at 500 PM pyruvate. In addition, Table I presents measurements of pyruvate consumption under these conditions, which gives a good approximation of the flux through the pyruvate dehydrogenase, as pyruvate carboxylase activity is very low in heart. It is seen that in the presence of pyruvate as sole substrate, flux through the pyruvate dehydrogenase reaction increases slightly with increasing pyruvate concentration and that this is reflected in a diminished O/pyruvate ratio. This suggests a lesser number of oxidative steps subsequent to pyruvate dehydrogenase in the metabolism of pyruvate present at the higher concentrations. In the presence of pahnitoylcarnitine (Table I), the flux through pyruvate dehydrogenase is greatly diminished, though appreciable at the higher pyruvate concentrations, when measured at 10 min. Inspection of the data for PDH, content revealed a rapid change during the first 5 min of incubation with palmitoylcarnitine, followed by a near steady state between 5 and 10 min. For this reason, pyruvate consumption during the first 5 min of these incubations was also measured, so that flux through pyruvate dehydrogenase for the period 5 to 10 min could be determined by subtraction and compared with the near steady state PDH, content for that time period. When this was done (Table I) it was found that flux through pyruvate dehydrogenase between 5 and 10 min of incubation was almost abolished by palmitoylcarnitine, and indeed was less than 20% of the V,,, activity of the PDH, present (bottom

measured (PM)

14.8

58.3 47.5 36.8 31.0 (f3.1) 44

14.0

47.8 35.9 29.5 27.8 (f4.2) 49

13.6

41.7 34.2 28.4 26.4 W.9) 50

46.7 2.63 20.6

17.7

49.9 3.17 17.6

15.8

15.1

250

47.9 3.17 16.6

100

as sole substrate

50

Pyruvate

63.4 61.2 56.1 52.2 (*4.4) 24

13.2

45.7 2.28 27.0

20.1

500

(22.7) (f0.07) (f0.7)

(kO.7)

41.7 23.8 17.1 W.9) 15

25.6 11.9 8.8 (f2.4) 20

19.6 9.4 (*::;,

52.6

3.0

55.8 12.5 6.2

4.6

250

40.6

2.1

54.2 25 2.5

2.3

109

plus pyruvate

34

0.2

54.8 47 2.2

1.2

50

Pahnitoyl-L-carnitine

fs 8 51.6 &3.3) 5

.I

e

61.6 55.6

(f4.2) (k2.3) (*0.4)

(f0.4)

8 b

2.9

50.0 6.4 13.7

8.3

500

63.8

(PM)

- . - ._” - - ._ -- .I -- - - -

u Mitochondria (1.5 mg of protein) were added to 2 ml of incubation medium comprising 0.12 M KCl, 20 mM potassium Hepes, 20 mM potassium phosphate, 0.5 mre L-malate, 1 mM ATP, 1 mM MgCb, and 2.5 mg/mI of bovine serum albumin. The pH was 7.2. In addition, either pyruvate or pyruvate plus pahnitoykcarnitine was present as oxidizable substrate, as shown in the table heading. The concentration of pyruvate was varied, as shown, but the concentration of palmitoyl-L-camitine was always 50 CM. The recording of 02 uptake, the addition of pyruvate during the experiments on the basis of 02 uptake, the sampling for, and measurement of, PDH, content, and the sampling for, and measurement of, pyruvate concentration are all described in the Experimental Procedures section, Incubations were terminated at either 5 or 10 mm and pyruvate consumption in 5 min was sub&rated from that in 10 min to give the value described as “flux through PDH, 10-5 mm.” Values are means of three determinations, each made with a different mitochondriai preparation, with the average SEM presented at the right side of or below, the appropriate column of numbers. The preparations used for the measurement of PDH. content were the same as those used for 10 min flux determinations.

Flux through PDH, 10-5 min as 8 of [PDH.], mean of 5- and lo-min values

Ox uptake, 10 min (ngatom/min/mg) O/pyruvate ratio, 10 min Flux through PDH, 5 min (nmol/min/mg) Flux through PDH, 10-5 min (nmol/min/mg) [PDHJ mnol/min/mg at: lmin amin 5min 10 min

(mnol/min/mg)

Flux through PDH, 10 min

Parameter

TABLE I FLUX THROUGH THE PYRUVATE DEHYDROGENASE REACTION AND THE CONTENT OF ACTIVE PYRUVATE DEHYDROGENASE (PDH,) DURING THE STATE 4 OXIDATION OF PYRUVATE OR PYRUVATE PLUS PALMITOYL-L-CARNITINE BY ISOLATED RAT HEART MITOCHONDRIA~

PYRUVATE

DEHYDROGENASE

CONTROL

the end product inhibition of pyruvate dehydrogenase by NADH and acetyl-CoA, which has been described for the purified enzyme (5-8), an adequate explanation of the difference between the values for PDH, content and for flux through pyruvate dehydrogenase obtained in the present study? Granted that mitochondrial NAD’ and NADH concentrations behave in reciprocal fashion, as do mitochondrial CoA and acetyl-CoA concentrations, and that metabolic changes tend to affect both ratios, probably all that can be done on this score is to look for possible anomalies in the direction of change of one or both of these ratios. On the first point, it is seen (Table IL) that whereas NAD’/NADH ratios are similar at 250 and 500 PM pyruvate in the presence palmitoylcarnitine, absence of EA/acetyl-CoA ratios are much lower in the presence of the acylcarnitine. Indeed,

line). By contrast, the flux through pyruvate dehydrogenase with pyruvate as sole substrate was approximately equal to 50% of the PDH, value, with the exception of the experiment at 500 j.kM pyruvate, where the proportion fell to 24%. These results can be interpreted at two levels with the help of information on mitochondrial and CoA/acetyl-CoA NAD+/NADH ratios under the same experimental conditions (Table II). First, is inactivation of pyruvate dehydrogenase (decrease in PDH, content) in the presence of palmitoylcarnitine invariably associated with a decrease in one, or both, of these CoA/acetyl-CoA and NAD’/ ratios? NADH ratios have previously been shown to affect steady state levels of PDH, both using the purified pyruvate dehydrogenasekinase-phosphatase system (11) and using intact mitochondria (15-18, 24). Second, is TABLE RATIOS

OF MITOCHONDRIAL

COASH/ACETYL-COA CONDITIONS

Parameter

measured

Mitochondrial CoASH content (nmol/mg of protein1 Mitochondrial acetyl-CoA content (mnol/mg of protein) Ratio CoA/acetyl-CoA Mitochondrial long-chain acylCoA content (nmol/mg of protein) Mitochondrial NAD+ content (nmol/mg of protein) Mitochondrial NADH content (nmol/mg of protein) Ratio NAD’/NADH

Pyruvate

69

IN HEART

II

AND NAD’/NADH OF TABLE I”

as sole substrate (PM)

UNDER THE EXPERIMENTAL

Palmitoyl-L-car&me plus pyruvate (pM)

50

100

250

509

50

0.48

0.40

0.18

0.12

ND*

ND

100

ND

250

ND

500

0.53

0.67

0.82

1.00

0.44

0.44

0.44

0.45

0.92 NM

0.59 NM

0.22 NM

0.12 NM

0.67

0.65

0.63

0.60

4.81

4.54

4.23

4.49

4.76

4.61

4.42

4.39

0.59

0.74

0.81

0.85

1.12

0.91

1.07

0.85 (kO.07)

8.2

6.1

5.2

5.3

4.3

5.1

4.1

5.2

(ltO.05)

n The protocol was basically that of the experiments presented in Table I, with the modification that 12 mg of mitochondrial protein was used for each incubation and the volume was 10 ml. The medium was saturated with C2 to avoid anaerobiosis and this precluded precise recording of 02 tension and necessitated supplementation with pyruvate on the basis of time elapsed, rather than 0~ consumed, as in Table I. In the experiments involving pahnitoylcamitine, an additional 200 nmol of palmitoyl-L-car&me was added at 6 mm to avoid depletion of this substrate. Two-milliliter samples were removed in quadruplicate between 7.5 and 8 min and were expelled into HClO, or ethanolic KOH for the measurement of NAD+ and CoA derivatives or NADH, respectively. Details of assay procedures are given in the Experimental Procedures section. The values given are the means of results obtained from two separate extracts, except for data on nicotinamide nucleotides in the experiments using palmitoyl-L-carnitine, where values are from four extracts using two different mitochondrial preparations. The errors shown are typical for nicotinamide nucleotide determinations; errors for CoASH and acetyl-CoA determinations are smaller (see Hansford (15,17)). Mean ATP/ADP ratio in these incubations was 30 f 2. b ND, not detectable. ’ NM. not measured.

70

HANSFORD

CoA was undetectable in these experiments, using a very sensitive kinetic assay (21). The lowered CoA/acetyl-CoA ratio is thus a plausible cause of the lowered PDH, content found in the presence of pahnitoylcarnitine (Table I). It should be noted that the ATP/ADP ratio, assayed in extracts of the whole mitochondrial incubation, was unchanged by the presence of the acylcarnitine and is therefore not a plausible modulator of the interconversion. On the second point, within the series of experiments with pyruvate as sole substrate, flux is constant at approximately 50% of the PDH, value at pyruvate concentrations of 50 to 250 PM and then falls to approximately 25% at 500 PM (Table I). There is no difference in NAD+/NADH ratio between experiments at 250 and 500 PM pyruvate (Table II) but there is a change in CoA/acetyl-CoA ratio. Thus, the increased feed-back inhibition apparent at 500 PM pyruvate is probably due to increased acetyl-CoA and decreased CoA, as the inhibition is competitive (7, 8), and not to any change in mitochondrial redox status. The generally much greater degree of feed-back inhibition seen in the presence of pahnitoylcarnitine (Table I) can similarly be attributed to the very low CoA/acetylCoA ratios in that state (Table II) but the reason for the difference in results between incubations with 250 PM and 500 ,UM pyruvate (plus palmitoylcarnitine) remains obscure. The possibility of direct inhibition of PDH, by the long chain acyl-CoA generated in the presence of pahnitoylcarnitine is discussed in a later section. Inactivation and feed-back inhibition of

pyruvate dehydrogenase due to palmitoylcarnitine oxidation in respiratory states between 3 and 4. In a previous paper (17), the merits of investigating metabolic states intermediate between 3 and 4 were discussed. Table III presents the results of such an experiment, in which the rate of respiration was limited by the rate of ADP provision and was adjusted to 50% of a state 3 rate (measured at saturating pyruvate concentration) by the careful addition of hexokinase. The respiratory state was characterized by rates of 02 uptake of approximately 170 ng-atoms of O/min/mg of protein ( cfi approximately 50 in Table I),

AND

COHEN

ATP/ADP ratios of approximately 19 (cf 30 in Table I) and values of PDH, content substantially higher than in state 4. This difference can probably be attributed to the effects of both the ATP/ADP and NAD+/NADH ratios on pyruvate dehydrogenase interconversion. Interconversion is sensitive to the former ratio because ADP inhibits the kinase (23) and because differential chelation of Mg+ by ADP and ATP affects kinase and phosphatase differently (25): it is sensitive to the latter ratio because NADH activates the kinase and NAD+ antagonizes this effect (11, 13). It is seen that, contrary to previous work (17), pahnitoylcarnitine oxidation gave rise to no decrease in PDH, content in this metabolic state (Table III). This discrepancy is addressed below. Because of this lack of pyruvate dehydrogenase inactivation, all of the diminution in flux through pyruvate dehydrogenase seen in the presence of palmitoylcarnitine (Table III) can be attributed to feed-back inhibition of PDH,. A major cause is probably the very low CoA/acetyl-CoA ratio seen with palmitoylcarnitine in other 50% state 3 studies (see below), though not measured here. Within the series of experiments with pyruvate as sole substrate (Table III), flux (measured only at 10 min, as PDH, content is stable with time) is almost invariant at approximately 55% of PDH, activity, except at 500 pM pyruvate, where it is appreciably higher. Since the CoA/acetyl-CoA ratio, which is measured with some precision as in earlier work (15, 17), is a minimum at 500 PM pyruvate, the increased activity of the PDH, at this substrate concentration may reflect either a decreased feed-back inhibition by NADH, though the small change in the NAD+/NADH ratio is not compelling or, more probably, an increased degree of saturation of Enzyme-l of the complex with its substrate, pyruvate. If this is true, it suggests that K,,, values for pyruvate of less than 100 ,UM determined for the isolated enzyme (8) may not apply to the enzyme in its intramitochondrial milieu, though this must remain a tentative conclusion. In the present studies, the pyruvate concentration in the mitochondrial matrix will probably be greater than that added to the medium, assuming equilibration of pyruvate distri-

measured

PDH, 10 min, as % of 5.min value

0.42 0.81 0.52 4.67 0.70 6.7

0.72 0.64 4.87 0.60 8.2

85.0 82.6 82.2 91.1 &6.4) 55

79.5 77.3 79.9 80.8 (*5.0) 53

0.46

171 3.75

170 4.0

6.6

0.66

0.28 4.34

0.91

0.25

89.5 88.5 88.9 92.8 (k7.7) 55

168 3.45

48.7

250

as sole substrate

45.3

100

42.4

50

(PM,

7.5

0.56

0.19 4.22

0.99

0.19

90.1 88.5 84.1 91.5 (f2.6) 72

171 2.80

60.4

500

(2% (+O.lO)

(f2.1)

UNDER THE SAME EXPERIMENTAL CONDITIONS* Pyruvate

NM

10.2 80.8 80.9 81.4 84.4 (f5.7) 21

13.6 73.8 72.8 73.7 78.3 (k5.5) 19

NM

17.4 177

14.2 184

106

Palmitoyl-L-carnitine 50

NM

(2% 23

83.6 87.4 87.5

8.7

19.8 171

250

plus pyruvate

(FM)

NM

82.2 80.2 85.1 90.3 (+3.2) 39

5.5

33.5 183

506

(*toi

(k1.7) (53

* Mitocbondria (0.6 mg of protein) were added to 2 ml of medium of the composition given in the legend to Table I, with the addition of 10 rnM ri-glucose. The concentration of pyruvate is given in the table. Immediately after the addition of the mitochondria, an amount of dialyzed hexokinase found in control O2 electrode studies to elicit a rate of 02 uptake of 50% of the state 3 rate was also added. Pyruvate supplementation during the course of the experiment was on the basis of O2 uptake (see Experimental Procedures section). Values represent the mean of three determinations, each with a different mitochondrial preparation; with the error being the average SEM for each group of data. *The protocol was basically as described above with the exception that 12 mg of mitochondrial protein and 10 ml of medium was used for each experiment. Oxygen was blown at the surface of the liquid to maintain an aerobic state, and this was monitored with an O2 electrode. Supplementation of pyruvate during the course of the experiment was on the basis of time elapsed. The value given is the mean of duplicate determinations, involving separate extracts. Mean ATP/ADP ratio in these incubations was 19.4 -C 0.2

0% Mitochondrial CoASH content (mnol/mg of protein) Mitochondrial acetyl-CoA content (rmiol/mg of protein) Ratio CoA/acetyl-CoA Mitochondrial NAIY content (nmoI/mg of protein) Mitochondrial NADH content (nmol/mg of protein) Ratio NAD’/NADH

Flux through [PDH.],

Flux through PDH, 10 min (mnol/min/mg) O2 uptake, 10 min (rig-atom/min/mg) O/pyruvate ratio, 10 min [PDH.] nmol/min/mg at: 1 min 2min 5min 10 mm

(-4)

Parameter

TABLE III (A) FLUX THROUGH THE PYRUVATE DEHYDROGENASE REACTION AND THE CONTENT OF PDH, DURING THE OXIDATION OF PYRUVATE OR PYRUVATE PLUS PALMITOYL-L-CARNITINE AT A RATE OF O2 CONSUMPTION OF 50% STATE 3” AND (B) RATIOS OF MITOCHONDRIAL COASH/ACETYL-COA AND NAD+/NADH

2

2

E

3

8

ii

2 z

g

3

8

z M

z

measured

37

79.3 76.0 70.4 65.9 (f2.1) 54

43

71.4 64.3 57.3

36

68.9 66.5 64.1 58.7 (k5.6) 58 (E, 79

157 3.57 51.2

151 3.45 43.8

150 3.55 43.3

250 44.1

(PM)

43.2

100

as sole substrate

39.4

50

Pyruvate

(2:) 50

72.2 76.2 74.2

35

154 2.95 66.4

50.6

(3zO.10) (k2.1)

W2)

(f3.5)

500

(k3.6)

(k2.6)

75.7 69.6 56.8

56.8 39.5 26.6 36.8 12.4 6.8 21.4 6.1 (2,

(jz2.0)

70.4

1.2

66.7

-

18.7 170 10 36.2

(phi)

50.4

1.4

-

174 19 20.2

7.1

250

plus Pyruvate

42.0

170 40 7.4

4.4

100

170 69 5.1

1.6

50

Palmitoyl-L-car&me

(k12) (f4) (k2.6)

(k1.9)

500

“The protocol was as described for Table IIIA with the sole exception that the medium also contained 0.5 mru EGTA. Values are the mean of three determinations, each made with a different mitochondrial preparation, with the error being given by the average SEM at each group of data. The same three preparations were used for the determination of PDH. content and of IO-min flux.

Flux through PDH, 10-5 min as 56 of [PDH,], mean of 5- and IO-min values

Flux through PDH, 10 min bmol/min/mg) O2 uptake, 10 min (ng-atom/min/mg) O/pyruvate ratio, 10 mm Flux through PDH, 5 min bmol/min/mg) Flux through PDH, 10-5 min (nmol/min/mg) [PDH.]mnol/min/mg at: lmin amin 5min 1omin

Parameter

TABLE IV FLUX THROUGH THE PYRUVATE DEHYDROGENASE REACTION AND THE CONTENT OF PDH, DURING THE OXIDATION OF PYRUVATE OR PYRUVATE PLUS PALMITOYL-L-CARNITINE IN AN EGTA-CONTAINING MEDIUM, AT A RATE OF O2 CONSUMPTION OF 50% STATE 3”

is Z

8

ti

E

PYRUVATE

DEHYDROGENASE

CONTROL

periment, the former dramatically (Table V), either of these parameters could be responsible for this enhanced inactivation. Although the lo-min flux through pyruvate dehydrogenase in the presence of palmitoylcarnitine was quite high at the higher pyruvate concentrations, subtraction of pyruvate disappearance occurring in the first 5 min revealed that flux between 5 and 10 min was essentially nil (Table IV). When compared with the quasi steady state values of PDH, at the same time points, this indicates a very great degree of feed-back inhibition, especially at 250 and 500 PM pyruvate. Inasmuch as CoA/acetyl-CoA and NAD+/NADH ratios are essentially invariant with pyruvate concentration in these (plus palmitoylcarnitine) experiments (Table V), the greater degree of feedback inhibition at the higher pyruvate concentrations is hard to explain, though it is conceivable that PDH, activity is responding to changes in CoA content which are below the limits of detection. In the experiments with pyruvate as sole substrate, the flux through the pyruvate dehydrogenase reaction is approximately constant as a proportion of the PDH, content, suggesting that, as in the experiment of Table III, increased end-product inhibition by acetylCoA is balanced by increased saturation of

bution with the ApH across the mitochondrial membrane (26). The most striking aspect of these experiments is probably the manner in which flux is maintained constant as the pyruvate concentration is raised from 50 to 250 PM by allowing the progressive and inhibitory decrease in the CoA/acetyl-CoA ratio. The only experimental variable which could be identified as not properly controlled in the previous work (17) and in the experiments of Table III was the concentration of divalent cations and, specifically, Ca2+, which was not added but would be present to some degree as a contaminant. Thus, the unexplained enhancement of pyruvate dehydrogenase inactivation in the previous study seen on adding ATP and MgCl2 in state 4 incubations could have conceivably involved a lowering of free Ca2+.With this in mind, the experiments of Table III were repeated in a medium differing only by the inclusion of 0.5 mu EGTA. The difference was dramatic, with a pronounced, and rather slow, inactivation of pyruvate dehydrogenase occurring in the presence of palmitoylcarnitine (Table IV), at all but the highest concentration of pyruvate. As both CoA/acetyl-CoA and NAD+/NADH ratios are decreased by the inclusion of pahnitoylcarnitine in this ex-

TABLE V RATIOS OF MITOCHONDRIAL COASH/ACETYL-COA AND NAD’/NADH CONDITIONS OF TABLE IV” Parameter

measured

Pyruvate

as sole substrate

UNDER THE EXPERIMENTAL

plus pyru-

Pahnitoyl-L-carnitine

(PM) Mitochondrial CoASH content (nmol/mg of protein) Mitochondrial acetyl-CoA content (mnol/mg of protein) Ratio CoA/acetyl-CoA Mitochondrial long-chain acyl-CoA content (runol/mg of protein) Mitochondrial NAD+ content (mnol/mg of protein) Mitochondrial NADH content (mnol/mg of protein) Ratio NAD’/NADH

73

IN HEART

vate (PM

50

loo

250

500

50

0.32

0.29

0.26

0.18

ND

0.93

0.80

0.88

0.81

0.40 NM

0.37 NM

0.29 NM

4.62

4.38

0.58 8.0

100

250

500

ND

ND

ND

0.23

0.23

0.25

0.30

0.23 NM

0.73

0.67

0.68

0.65

4.28

3.51

5.08

4.67

4.45

4.51

0.51

0.56

0.43

0.69

0.63

0.67

0.64

8.6

7.7

8.4

7.3

7.4

6.7

7.1

(1The protocol was as described for Table IIIB, with the exception that the medium contained 0.5 mM EGTA. In addition, pahnitoyl-L-car&me (250 nmol) was added at 3.5 and 7 min in appropriate incubations, to avoid depletion. The mean ATP/ADP ratio determined was 17 + 1 for experiments with pyruvate as sole substrate and 16 f 1 for experiments with pyruvate plus palmitoyl-L-carnitine. Values are the means of duplicate determinations, using separate extracts.

74

HANSFORD

Enzyme-l of the complex (pyruvate dehydrogenase) with its substrate pyruvate. There was no appreciable variation of mitochondrial NAD’/NADH ratio with pyruvate concentration in this experiment (Table V). The striking effect of EGTA, and by implication Ca2+, in these two experiments is analyzed more closely in a later section. Inactivation and feed-back inhibition of pyruvate dehydrogenase during the state 4 oxidation of pyruvate and pyruvate plus palmitoylcarnitine, in the presence of dichloroacetate. One object of the present work was to determine whether changes in flux through pyruvate dehydrogenase, as a proportion of the content of PDH,, were at least qualitatively in keeping with changes in CoA/acetyl-CoA and NAD+/NADH ratios. In the experiments described so far, pyruvate concentration was a major determinant of PDH, content and, in the experiments also involving pahnitoylcarnitine, of flux through pyruvate dehydrogenase. However, the substrate role of pyruvate makes the end-product inhibition difficult to analyze. For this reason, a study was carried out in which PDH, content was varied by adding dichloroacetate (Table VI), a nonmetabolizable carboxylate which has been shown to inhibit pyruvate dehydrogenase kinase (27). It is seen that in the presence of 50 PM pyruvate as sole substrate, addition of dichloroacetate to concentrations of up to 500 pM raised the steady state content of PDH,. It is not clear why higher concentrations were necessary to cause an effect than in the original studies of Whitehouse et al. (27). Possibly the higher concentration of Pi in the present work is responsible for a diminished ApH across the mitochondrial membrane and a consequently lower concentration gradient of dichloroacetate. When flux is expressed as a proportion of PDH, content, it declines with increasing dichloroacetate. At the same time, there is a decrease in NAD+/NADH ratio and a 20fold decrease in CoA/acetyl-CoA ratio (Table VII). Clearly, PDH, in its intramitochondrial milieu cannot be more than very slightly sensitive to end-product inhibition by acetyl-CoA. It is notable that Bremer (7) found a much lesser inhibition

AND

COHEN

by acetyl-CoA than by NADH when purified kidney pyruvate dehydrogenase was incubated with mixtures of CoA and acetylCoA and of NAD+ and NADH, of constant total molarity, but varying proportion. In the series of experiments with both 50 PM pyruvate and palmitoylcarnitine as substrates (Table VI), flux is a constant proportion of PDH, content, within the large errors implicit in the subtraction of values of pyruvate uptake determined with different mitochondrial suspensions (albeit mean values of triplicate experiments). This criticism really only applies to state 4 experiments with pahnitoylcarnitine, where the flux through pyruvate dehydrogenase is always very small. Nevertheless, this approximate constancy of flux as a proportion of PDH, content (Table VI) is in keeping with the unchanged NAD’/NADH and CoA/acetyl-CoA ratios seen on increasing dichloroacetate concentration in the experiments with palmitoylcarnitine (Table VII). Inactivation and feed- back inhibition of pyruvate dehydrogenase during the state 4 oxidation of 100 PM pyruvate in thepresence of different ratios of n,L-carnitine/acetyl-o,L-carnitine. Finally, an experimental protocol was developed in which pyruvate concentration was kept constant, to avoid changes in enzyme activity with the degree of saturation with substrate, and in which the CoA/acetyl-CoA ratio was varied systematically using acetyl-n,L-carnitine as acetyl-group donor and D,L-carnitine as acceptor (Table VIII). This allowed smaller changes in this ratio than were possible in previous experiments with palmitoyl-L-carnitine as substrate. It was hoped that it might be possible to evaluate feedback inhibition by acetyl-CoA alone. It is seen that flux through pyruvate dehydrogenase increases as the ratio D,Lcarnitine/acetyl-n,L-carnitine increases. Indeed, the O/pyruvate ratios show that a change occurs from a situation in which acetylcarnitine donates acetyl-groups for oxidation by the tricarboxylate cycle (column 1) to one in which carnitine accepts almost all of the acetyl groups formed from pyruvate, especially in 5-min incubations (column 6; O/pyruvate = 1.02 + 0.03 at 5 min, not shown). When flux is expressed as a proportion of PDH,, it can be seen that it

TABLE

VI

63 58 67 47 19

55 52 54 41 26

46 40 71 57 18

76 70

11.5

3.17 11.3

36.7

11.4

(kO.14) (kO.9)

(A4.8)

(k1.2)

24 16 8

39 32

1.7

40 2.3

39.7

2.0

30 21 4

50 42

1.1

32 2.1

41.6

1.6

45 34 7

2.7

2.8

37 23 9

16 3.7

16 2.4

74 66

42.4

36.8

62 52

3.2

2.6

(r5.6)

(zkO.9)

59 47 9

78 77

4.7

10 (*lo) 4.1 (kO.4)

46

4.4

a Mitochondria (1.5 mg of protein) were added to 2 ml of incubation medium comprising 0.12 M KCl, 20 mM potassium HEPES, 20 mM potassium phosphate, 0.5 mM b-malate, 0.5 mM EGTA, 2.5 mg/ml of bovine serum albumin, and 50 PM pyruvate. In addition, the medium contained the concentration of dichloroacetate shown above and, where indicated, 50 PM palmitoyl-L-carnitine. Supplementation with pyruvate during the experiments and sampling and assay techniques are described in the Experimental Procedures section. The value given is the mean of three determinations, each using a different mitochondrial preparation.

46 36 29

12.5

12.4

11.6

3.20 9.3

3.27 9.0

mm

35.0

34.7

33.0 3.43 7.4

10.9

10.7

9.5

min

5 min 10 min Flux through PDH, 10-5 min, as % of [PDHJ, mean of 5- and lo-min values

O/pyruvate ratio, 10 mm Flux through PDH, 5 (nmol/min/mgl Flux through PDH, 10-5 (nmol/min/mg) [PDH.] nmol/min/mg at: 1 min 2 min

Flux through PDH 10 min (nmol/min/mg) Ox uptake, 10 min (ng-atom/min/mg)

9

FLUX THROUGH THE PYRLJVATE DEHYDROGENASE REACTION AND THE CONTENT OF PDH. DURING THE STATE 4 OXIDATION OF 50 PM PYRUVATE AND 50 PM PYRUVATE PLUS PALMITOYLCARNITINE IN THE PRESENCE OF VARIOUS CONCENTRATIONS OF DICHLOROACETATE” Concentration of dichloroacetate Pyruvate Pyruvate plus palmitoyl-L-carnitine (UM) 250 500 1ooo 100 250 500 NIL 100 NIL

d

3

2

E

2

8

z B3

$

3

I2

ti

2

5

76

HANSFORD

AND

COHEN

TABLE VII RATIOS OF MITOCHONDRIAL COASH/ACETYL-COA AND NAD+/NADH CONDITIONS OF TABLE VI” Substrate Concentration

of dichloroacetate (PM)

Mitochondrial CoASH content (mnol/mg of protein) Mitochondrial acetyl-CoA content (nmol/mg of protein) Ratio CoA/acetyl-CoA Mitochondrial long-chain acyl-CoA content (nmol/mg of protein) Mitochondrial NAD+ content (nmol/mg of protein) Mitochondrial NADH content (mnol/mg of protein) Ratio NAD’/NADH

UNDER THE EXPERIMENTAL Pyruvate

Pyruvate

plus pahnitoyl-L-carnitine

NIL

100

250

500

NIL

0.90

0.73

0.28

0.12

ND

0.25

0.44

0.58

0.74

3.6 0.19

1.7 0.19

0.48 0.21

4.64

4.19

1.12 4.1

100

250

500

ND

ND

ND

0.26

0.24

0.26

0.26

0.16 0.21

0.52

0.54

0.64

0.57

4.15

3.97

4.47

4.13

3.88

3.72

1.26

1.32

1.53

1.49

1.43

1.32

1.19

3.3

3.1

2.6

3.0

2.9

2.9

3.1

a The protocol was basically as described for Table VI, with the exception that each incubation contained 12 mg of mitochondrial protein in a volume of 10 ml. Supplementation with pyruvate and sampling techniques were as described for Table II. Extra pahnitoyl-L-car&me (200 nmol) was added at 6 min in appropriate incubations to avoid depletion. The value given is the mean of duplicate determinations using separate extracts.

increases with the D,L-carnitine/acetyln,L-carnitine ratio. In the absence of changes in pyruvate concentration (see Experimental Procedures section), this can be attributed to a decreased feed-back inhibition by acetyl-CoA. It is noteworthy that the change in the CoA/acetyl-CoA ratio determined for this set of experiments is very large (Table IX), but that the diminished inhibition due to this change must be offset to some extent by a change of the NAD+/NADH ratio in the opposite direction. Thus, again no quantitative conclusions about inhibition by acetyl-CoA are possible. At the level of pyruvate dehydrogenase interconversion, it is seen that the final steady state content of PDH, varies only very slightly with the ratio D,L-carnitine/acetyl-n,L-carnitine (Table VIII). Inspection of Table IX allows the statement that a decrease in NAD+/NADH ratio from 3.5 to 2.2 essentially matches an increase in CoA/acetyl-CoA ratio from 0.67 to 12 in the modulation of pyruvate dehydrogenase interconversion. Thus, interconversion is much more sensitive to changes in redox state than to changes in CoA acetylation. Experiments on pyruvate deh?drogenase interconversion using Ca +-EGTA buglers. The discrepancy between results

obtained in experiments which included the chelating agent EGTA (Table IV) and those which did not (Table III), focused attention on the importance of Ca2+ concentration in modulating the steady state level of PDH,. An effect of Ca2+ions, present at micromolar concentrations, in activating pyruvate dehydrogenase phosphatase was shown by Denton et al. (28) and was established to involve a change in the apparent K, of the phosphatase for phosphorylated pyruvate dehydrogenase (29, 30). Subsequently, Severson et al. (31) showed that treatment of isolated fat cell mitochondria with the divalent-metal ionophore A23187 led to inactivation of pyruvate dehydrogenase phosphatase, which could be reversed by the addition of both MgC12 and CaC12.Similar results were obtained by Walajtys et al. (32), who used isolated liver mitochondria. However, studies in which fat cell mitochondria were incubated with EGTA, but no ionophore, gave no appreciable diminution in phosphatase activity, even though the mitochondrial Ca2+ content could be shown to be diminished by 60% (31). With this in mind, it was thought worthwhile to investigate further the present findings, which involved incubations of mitochondria with EGTA but without A23187. Results from a pre-

PYRUVATE

DEHYDROGENASE

77

CONTROL IN HEART

TABLE VIII FLUX THROUGH THE PYRUVATE DEHYDROGENASE REACTION AND THE CONTENT OF PDH, DURING THE STATE 4 OXIDATION OF 100 PM PYRUVATE IN THE PRESENCE OF VARIOUS CONCENTRATIONS OF ACETYL-D,L-CARNITINE AND D,L-CARNITINE~

Parameter measured

Ratio of D,L-carnitine/acetyl-D,L-camitine (ii:;,

Flux through PDH, 10 min (nmol/min/mg) 02 uptake, 10 min (ng- atom/min/mg) O/pyruvate ratio, 10 min Flux through PDH, 5 mm (nmol/min/mgl Flux through PDH, 10-5 min (nmol/min/mg) [PDH,] nmol/min/mg at: lmin 2min 5min

10 min

(l%,

2%

mM)

(lT5,

3.9

5.4

7.5

8.6

9.4

12.1 (jzO.4)

20.5

19.7

18.5

17.4

17.9

20.1 (kO.8)

5.6 5.9

3.7 10.7

2.5 12.0

2.1 13.6

1.9 14.9

1.7 (kO.27) 17.7 (k1.9)

3.6

3.6

3.9

6.5

53.0 46.8 31.8 30.0 (lt1.8) 10

58.8 54.9 37.7 31.6 (k3.4) 10

63.1 53.3 38.2 33.2 (k2.5)

63.1

(k2.9)

11

17

1.9

43.9

39.1 26.1 25.7 (jz2.1)

Flux through PDH, 10-5 min, as % of [PDH.], mean of 5- and IO-min values

(it)

(total concentration,

7

49.9 43.5 29.5 29.1 (f1.3) -

59.5 42.9 32.1

n Mitochondria (1.5 mg of protein) were incubated in 2 ml of a medium of composition the same as that given for Table VI, but with the exception that the pyruvate concentration was 100 m instead of 50 ,a~. Instead of dichloroacetate, mixtures of D,L-carnitine and acetyl-n,L-camitine were present in each incubation and were of the proportions and total concentrations indicated, such that one calmpound, on both, was present in each incubation. Supplementation with pyruvate during the experiments *and sampling and assay techniques are described in the Experimental Procedures section. Values are means of four determinations, each made with a different mitochondrial preparation, with the error being the average SEM for each group of data.

TABLE

IX

RATIOS OF MITOCHONDRIAL COASH/ACETYL-COA AND NAD+/NADH CONDITIONS OF TABLE VIII”

Parameter measured

.Mitochondrial CoA content (nmol/mg of protein) Mitochondrial acetyl-CoA content (nmol/mg of protein) Ratio CoA jacetyl-CoA Mitochondrial long-chain acyl-CoA content (nmol/ml of protein) Mitochondrial NAD+ content (mnol/mg of protein) Mitochondrial NADH content (nmol/mg of protein) Ratio NAD’/NADH

UNDER THE EXPERIMENTAL

Ratio of n,L-carnitine/acetyl-n,L-camitine

(total concentration,

(&

(l%)

0.62

095

1.31

1.41

0.93

0.63

0.33

0.12

0.67 0.08

I.!5

4.0

0.08

0.06

12 0.07

4.24

3.'71

3.18

3.30

1.19

1.47

1.66

1.48

(lF5,

1.9 2.2 n Mitochondria were incubated under four of the six experimental conditions outlined in Table VIII, but using 12 mg of protein and a volume of 10 ml. Results are the mean values of determinations using two separate extracts. 3.5

3.0

78

HANSFORD AND COHEN

vious study of rabbit heart mitochondria by Chiang and Sacktor (33), though provocative, could not be directly related to the present work, as nonrespiring mitochondria and low-salt incubation media were used. Respiration will affect the distribution of Ca2+ across the mitochondrial membrane because of the generation of a membrane potential (34, 35) and monovalent cations affect the activity of pyruvate dehydrogenase kinase (36). When the steady state content of PDH, was determined in the presence of 100 pM pyruvate and palmitoylcarnitine as substrate and at an intermediate rate of oxidative phosphorylation (50% state 3), the dependence upon free Ca2’ ion concentration shown in Fig. 1 emerged. Ca2+ ion concentrations were stabilized by using mixtures of CaC12 and EGTA as described by Portzehl et al. (37). It is seen that pyruvate dehydrogenase activity is much greater at pCa2+ = 6.2 than at pCa2+ = 6.9. Although EGTA is highly specific for Ca2+, some chelation of Mg2+ will occur in those experiments in which there is a large molar excess of EGTA over CaCl2 (37). As Mg2+ ions are required for phosphatase activity (9, lo), it was essential to show that the decreased steady state content of PDH, in the incubation of high pCa2+ did not reflect depletion of mitochondrial Mg2+. This is not so, as shown by the similar results (obtained with another mitochondrial preparation) obtained when MgC12 was added to titrate the EGTA present in excess of the CaC12 concentration (Fig. 1). Any possible physiological role of Ca2’ in modulating pyruvate dehydrogenase activity requires that the activation be reversible. Fig. 2 shows the results of triplicate experiments in which the free Ca2+ ion concentration of a mitochondrial incubation was first lowered with EGTA, then raised by the addition of a CaClJEGTA buffer and then finally lowered again by the addition of excess EGTA. The respiratory state was the same as in Fig. 1, so that the pyruvate dehydrogenase interconversion system was poised between extremes. This is important, as repetition of these experiments in the absence of palmitoylcarnitine expressed no modulation of PDH interconversion by pCa2+ (not shown). It is seen

%.6 I

6.0

6.2 I

6.4

6.6 ,

6.6

7.0

7.2 I

pCa” FIG. 1. The effect of free [Ca”‘] on PDH, content during the oxidation of pyruvate phs palmitoyl-L-barnitine by mitochondria respiring in 50% state 3. Mitochondria (0.6 mg of protein) were added to 2 ml of medium comprising 0.12 M KCl, 20 mM potassium Hepes, pH 7.2, 20 mM potassium phosphate, pH 7.2, 100 PM pyruvate, 0.5 mM L-malate, 0.9 mru ATP, 0.9 mM MgClz, 10 IIIM glucose, and 2.5 mg of bovine serum albumin/ml. In addition, mixtures of EGTA and CaCb were present to stabilize the concentrations of free Ca2+ shown (presented as the negative logarithm of the concentration, in molarity, or pCa*+). The final EGTA concentration was 2 mrvr.Dialyzed hexokinase was added to generate 50% of a state 3 rate and 02 uptake was recorded. Sampling for PDH, was at 10 min and was in duplicate. Rates of 02 uptake were unaffected by [Ca”], except in the experiment where pCa*+ = 5.8, where there was a slight increase. For the six other experiments, rates were in the range 137-143 ng-atoms of O/min/mg of protein. O--O, no further additions; M, MgClz was added to each incubation to a concentration equal to that of EGTA not bound to Ca*+. A different mitochondrial preparation was used.

(Fig. 2) that PDH, content increases rapidly (tM c 1 min) on raising the free Ca2+ ion concentration and then falls more slowly (tY2 = 3.5 min approximately) when EGTA is added. Thus, the variation of extramitochondrial Ca2+ ion concentration in the range of pCa2+ 6-7 does affect the balance of pyruvate dehydrogenase kinase and phosphatase activities, in the absence of the ionophore A23187 (cf Severson et al. 31).

PYRUVATE

DEHYDROGENASE

CONTROL

IN HEART

79

(and, strictly immeasurable) with palmitoylcarnitine than with any other substrate, for instance 5 mM acetyl-n,L-carnitine (Table IX), and thus it may not be necessary to postulate any such inhibition. The literature on this question is equivocal, with Garland and Randle (5) finding no inhibition of the purified pyruvate dehydrogenase complex by long-chain acyl-CoA, but with Erfle and Sauer (38) providing evidence for 00 2 4 6 6 10 12 14 16 18 20 inhibition of the closely related cw-ketoglutarate dehydrogenase complex. ButterTIME lmlnl worth et al. (39) report inhibition of the FIG. 2. The time-course and reversibility of the dihydrolipoyl transacetylase component of effect of Ca** on the steady state PDH. content of kidney pyruvate dehydrogenase by longmitochondria respiring in 50% state 3. Mitochondria chain acyl-CoA, in studies using microgram (0.9 mg of protein) were added to 2 ml of the medium quantities of enzyme protein in the cuvette. described for Fig. 1, but containing 0.5 mM EGTA It is our opinion that these findings do not instead of the Ca2+ buffer. Immediately thereafter, necessarily imply that inhibition occurs in sufficient samples were removed at the times shown the mitochondrion, where the concentrafor the estimation of PDH. content. Five minutes after adding the mitochondria, 250 d of a neutral (pH 7.2) tion of protein is vastly higher, providing Ca2+ buffer was added, containing 10 amol of EGTA potential binding sites for long-chain acyland 9.5 qol of CaC12: at 10 min, a further 10 pmol of CoA and lowering its effective concentraEGTA was added. The computed pCa*+ values for the tion. The variable degree of feed-back in5-lo- and lo-20-min segments of the experiment are hibition in the present studies suggests that 6.24 and,approximately 7.2, respectively. Values plotthe constant relation between pyruvate oxted are means + SEM of three separate incubations idation and PDHa content observed by Hilusing the same mitochondrial preparation. In one intunen and Hassinen in intact heart (19) cubation (0- - -0), a further addition of palmitoyl-Lmay have been a special case. In general, a carnitine (50 nmol) was made at 8 and 16 min to avoid possible substrate depletion. For this reason, the other correlation between increased feed-back invalues (u) from lo-20 min are the means of hibition and decreased CoA/acetyl-CoA raduplicate experiments only. tios is readily observed (Tables I, II, 250 and 500 PM pyruvate; Tables VI, VII). Interestingly, a decrease in CoA/acetyl-CoA DISCUSSION ratio can however be compensated for by a Feed-back inhibition of pyruvate dehy- higher pyruvate concentration (Tables III, drogenase. The use of a wide range of ex- IV, pyruvate as sole substrate), giving an perimental conditions and the comparison unchanged flux through pyruvate dehydroof flux through pyruvate dehydrogenase genase as a proportion of PDH, content. with PDH, content has allowed the evalu- This raises the question of what the Km of ation of the degree of feed-back inhibition the pyruvate dehydrogenase for pyruvate to which the dehydrogenase is subject in its really is in the mitochondrial milieu (see intramitochondrial milieu and of the con- above). In conclusion, although very large tribution of acetyl-CoA and NADH to that changes in CoA/acetyl-CoA ratio are reinhibition. Specific experiments have been quired to inhibit pyruvate dehydrogenase interpreted above and, in this section, only materially (Tables VI and VII, pyruvate as a few general comments will be made. First, sole substrate), such changes do occur the degree of feed-back inhibition is very (Tables VI and VII, pyruvate as sole subvariable but is always extremely high in the strate; Tables VIII and IX). By contrast, presence of palmitoylcarnitine. This sug- NAD+/NADH ratios are more stable, in gested that long-chain acyl-CoA might also keeping with their linkage to phosphate have a role in inhibition and this was mea- potential (40) and experimental conditions sured in Tables II, V, VII, IX. However, may be found in which the effect of this CoA/acetyl-CoA ratios were always lower ratio on pyruvate dehydrogenase inhibition

80

HANSFORD

is overriden by changes in the CoA/acetylCoA ratio (Tables VIII\and IX). Inactivation of pyruvate dehydrogenase by interconversion. Although in many cases both CoA/acetyl-CoA and NAD+/NADH ratios change in the same sense and their relative contributions to enzyme inactivation by interconversion cannot be assessed, in other cases some such discrimination is possible. Thus, in Tables IV and V, there is probably no difference in redox state between experiments with or without palmitoylcarnitine, at the lower pyruvate concentrations, and at either pyruvate concentration differences in PDH, content can probably be attributed to differences in CoA/acetyl-CoA ratio. Interestingly, the increased CoA/acetyl-CoA ratio essentially balances the decreased NAD+/NADH ratio, as the camitine/acetylcarnitine ratio is raised (Tables VIII and IX). Thus, PDH, content remains essentially constant. This contrasts with the decreased degree of endproduct inhibition discussed above and allows the statement that changes in NAD+/NADH ratio are more important vis-k-vis changes in CoA/acetyl-CoA ratio in modulating enzyme interconversion than in effecting feed-back inhibition. Clearly, changes in CoA/acetyl-CoA and NAD’/NADH ratios are only responsible for fine-tuning the pyruvate dehydrogenase interconversion. Effects of fatty acid oxidation on PDH, content are not seen either in the presence of very low ATP/ADP ratios (17) or in the presence of Ca” ions at concentrations above 10m6 M. Whereas it is considered that physiologically the ATP/ADP ratio probably changes minimally in heart muscle (19, 41), changes in Ca” ion concentration may be more significant. The demonstration that the steady state PDH. content responds reversibly to extramitochondrial Ca2+ ions in the range of pCa 2+ 6-7 (Fig. 2) adds nothing to what is known mechanisticahy of the interaction of Ca2+ ions with the phosphatase (28-30) but does raise the possibility that pyruvate dehydrogenase responds to changes in cytosolic free Ca2+ of this order. It is noteworthy that the concentration dependence of the effect of Ca2+ (Fig. 1) is very similar to that seen with the adenosine triphosphatase of the cardiac actomyosin-tropomyo-

AND

COHEN

sin-troponin system (42), the skeletal muscle phosphorylase b kinase (43) and the glycerolphosphate dehydrogenase of blowfly flight muscle mitochondria (44). By contrast, variation of Ca2+ concentrations below 10e6 M caused no variation in the rate of mitochondrial O2 uptake (Fig. 1, legend) so that an indirect effect of Ca2+ on pyruvate dehydrogenase interconversion involving an energy demand of Ca2+ uptake and increased NAD+/NADH and ADP/ATP ratios becomes unlikely. However, the possibility of such an indirect mechanism would have to be entertained if Ca2+ concentrations above 10m6M were used. Very recently, a report appeared by Portenhauser and Wieland (45) in which the effect of pahnitoylcarnitine and 2-oxoglutarate on PDH, content in rat heart mitochondria was described. Pahnitoylcarnitine was found to decrease the PDH, content in both states 3 and 4. In addition, flux through pyruvate dehydrogenase was measured, and the importance of end-product inhibition, especially in state 4 studies with palmitoylcarnitine, was established. The present results are in substantial agreement with those of Portenhauser and Wieland (45), but examine pyruvate dehydrogenase inactivation and inhibition over a wider range of conditions and, through the measurement of CoA and acetyl-CoA content, allow comment on the importance of the CoA/acetyl-CoA ratio, as well as the NAD+/NADH ratio, in determining enzyme activity. In addition, since the submission of this paper, a report appeared by Olson et al. (46) focusing attention on the importance of the concentration of CoA rather than the magnitude of the CoA/acetyl-CoA ratio in controlling pyruvate dehydrogenase activity and in mediating the response to fatty acids. The present paper, though clearly segregating the effects of CoA and acetyl-CoA on interconversion from those on feed-back inhibition, does not allow comment on whether it is absolute concentrations or the ratio which is involved, as in experiments with intact mitochondria concentrations of CoA and acetyl-CoA tend to be reciprocally related. ACKNOWLEDGMENT We thank Dr. Bertram Sacktor throughout this work.

for his interest

PYRUVATE

DEHYDROGEN

REFERENCES

ASE CONTROL

IN HEART

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