The inactivation of pyruvate dehydrogenase by fatty acid in isolated rat liver mitochondria

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE INACTIVATION OF PYRWATEDEHYDROGENASE BY FATTY ACID IN ISOLATEDRAT LIVER MITOCHONDRIA* Joseph J. Batenburg and Merle S. Olson Department of Biochemistry, College of Medicine, University of Arizona, Tucson, Arizona 85724 Received

July

21.1975

The influence of fatty acid on the interconversion of the pyruvate dehydrogenase complex (PDH) between its active (dephospho-) and inactive (phospho-) forms and on the intramitochondrial ATP/ADP, NADH/NAD and acetylCoA/CoASH ratios was studied in isolated rat liver mitochondria. Conditions were foyd in which the PDH activity was inversely correlated only with the NADH/NAD ratio. Under other conditions the PDH activity was inversely correlated solely with the acetyl-CoA/CoASH ratio. These experiments suggest that the activity of the regulatory enzymes involved in the inactivation and reactivation of the pyruvate dehydrogenase multienzyme complex may be controlled by both the intramitochondrial NADH/NAD+and acetyl-CoA/CoASH ratios. Since the discovery for the regulation the delineation reactions Various

of the activity of factors

involved reports

monovalent

in this

cations

mitochondria perception

in intact

(4,13,14) into

mitochondrial

tissues

are fatty

in the differential (phosphatase)

and di-

and

regulation

of

of the multienzyme

the mechanism of this

the fatty

(10-12)

and more recently

acids and ketone bodies.

PDH has been provided

suggested that

acid effect

fatty until

acid-mediated very recently.

PDH, pyruvate

o 19 75 by Academic Press. inc. of reproduction in o~v form reserved.

in isolated

Little,

debydrogenase

533

complex

if any,

inactivation

of the

Taylor --et al.

may be mediated by an alteration

by USPHSGrant HL-16111

+Abbreviation: Cop.vright All rights

(4-6)

adenine nucleotides

and activation

consideration.

Among the agents which have been shown to lead to an inactivation

of the PDH activity

*Supported

that

(l-3)

the PDH kinase and phosphatase

mechanism has become a crucial

(7-9) may be involved (kinase)

mechanism

of PDH' by Reed and his colleagues

which may regulate

have indicated

the inactivation complex.

of the phosphorylation-dephosphorylation

(14) in

Vol. 66, No. 2, 1975

oxidation-reduction

BIOCHEMICAL

state

of the mitochondria

mental evidence to substantiate were performed acid-mediated

to attempt inactivation

AND BIOPHYSICAL

this

to delineate

but presented

suggestion. a possible

of the mitochondrial

RESEARCH COMMUNICATIONS

The present

little

experi-

experiments

mechanism for the fatty PDH.

METHODSAND MATERIALS Rat liver mitochondria were prepared using a slight modification of the procedure of Schneider and Hogeboom (15). The homogenization medium contained 225 u&l mannitol, 75 n&i sucrose and 0.1 mM EGTA. The washing medium contained 225 mM mannitol and 75 mM sucrose. Mitochondrial protein was estimated using a biuret procedure (16). Mitochondria (7-10 mg protein) were incubated for 10 min at 25'C in 2 ml of a medium containing as standard components: 15 mMKC1, 2 mMEIYCA, 5 mM M&l , 50 mM Tris-Cl (pH 7.5), 10 mM potassium phosphate, 10 mM succinate, 30 &I glucose, 0.2 mM ADP, 20 u hexokinase (EC 2.7.1.1), 22.5 mM mannitol, 32.5 m&lsucrose and 0.16 g Dextran T40. The incubations were performed in 25 ml Erlenmeyer flasks in a shaking water bath. When PDH activity was to be assayed, the incubation was stopped with a rapid freezing technique essentially as described by Taylor et al. (14). The incubation mixture (0.5 ml) was pipetted into a tube containGga.5 ml ice-cold This mixture was immediately plunged into 2 mM dithiothreitol in 2% ethanol. a dry ice-acetone bath and kept in this bath for at least 10 min. After thawing in chipped ice the contents of the tube (or 0.25 ml ghereof; see legend to Table II) were transferred to 25 ml Erlapeyers kept ai 0 C. PDH act:? was assayed by measuring the formation of CO2 from [lCl pyruvate. was initiated by adding 1 ml of an assay mixtye containing: 5 mM ass5!% [lCl pyruvate (spec.act. 20 mC/mole), 1.11 mM NAD , 0.37 mM CoASH, 0.8 mM thiamine pyrophosphate, 5.2 mM cysteine, 5 mM oxaloacetate and 0.02% Lubrol wx. The flasks were closed with rubber serum stoppers equipped with plastic center wells and were shaken at 25'C in a water bath. After 10 min the i cubation was terminated by injection of 1 ml of 1% HC104 into the flasks. lr: CO2 was measured as described previously (8). For the assay of intramitochondrial ATP, ADP and NAD+, 1.0 ml of the incubation mixture was equally divided in 2 Eppendorf centrifuge tubes (1.5 ml), each containing 0.2 ml 15% HC104 and 0.5 ml silicone oil (spec.gr. 1.054). After centrifugation for 1 min at maximum speed in an Eppendorf microcentrifuge, the top layers were removed. Of the combined HC104 extracts 0.3 ml was neutralized with KOH. ATP, ADP and NAD+were assayed fluorometrically (17) in the neutralized extract. Intramitochondrial ATP and ADP concentrations were corrected for extramitochondrial adenine nucleotides retained with the mitoThe volume of extramitochondrial fluid chondria during the centrifugation. adhering to the mitochondria $ying the centrifugation was obtained from parallel incubations with [U- C] sucrose. As the incubation mixture contained hexokinase, which could exert its action during the centrifugation, it was considered useless to determine ATP and ADP in the upper layer after the centrifugation. Extramitochondrial adenine nucleotides were determined by in total extract minus intramitochondrial difference, i.e. adenine nucleotide The total mitochondrial extract was obtained by adding adenine nucleotide. 1 ml of ice-cold 1.2 N HC104 to the incubation mixture; the protein was removed by centrifugation; neutralization was accomplished with KOH.

534

5

Vol. 66, No. 2

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AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

For determination of NADH in the mitochondrial incubation 0.5 ml of incubation mixture was added to a centrifuge tube containing G.25 ml of l.N KOH in 10% ethanol (17). After neutralization with HClO to pH 8.5 and removing the protein and KC104 by centrifugation, NADHwas I!?/ etermined immediately. For assay of CoASHand acetyl-CoA, total extracts were obtained by adding 1 ml of ice-cold 1.2 N HC104 to the mitochondrial incubations; the protein was removed by centrifugation; neutralization was accomplished with KOH. To the extract 1 mM dithiothreitol was added and CoASHwas measured fluorometrically with a-ketoglutarate dehydrogenase (18). Acetyl-CoA was measured in the same cuvetteby adding phosphotransacetylase (EC 2.3.1.8) (18). All intermediate determinations and PDH assays were performed in triplicate and the results reported represent the average of the three values. &Ketoglutarate dehydrogenase was isolated from pig heart according to Sanadi --et al. (19). Hexokinase (type V) was obtained from Sigma, lactate dehydrogenase (EC 1.1.1.27) from Worthington and the other enzymes from Boehringer. Lithium acetoacetat~l~~~c~~~~~~~e~c~~~~~~s~r~~~ @&.i~~;t; T40 was supplied by Pharmacia. Omnifluor were obtained from New England Nuclear. Serum stoppers and center wells were supplied by Kontes. RESULTSAND DISCUSSION Addition has

of fatty

acids to metabolic

preparations

been shown to lead to an inhibition

nation

for this

effect

centered

and I?ADH (both products inhibitors

of PDH (21-23).

to intact

of PDH (4,10-14).

entirely

of pyruvate

around the fact

oxidation

Recently

metabolizing The early

and S-oxidation)

of the active

expla-

that both acetyl-CoA

it has been shown that

systems leads to a conversion

pyruvate

are competitive fatty

acid addition

to the inactive

form of

PDH (4,10-14). It

is an interesting

the PDH reaction

possibility

that

(NADH and acetyl-CoA)

mediated regulation

of the multienzyme

the known allosteric

may be involved complex.

such effects

Taylor -et al. PDH in rat bation state

liver

(14) recently mitochondria

conditions

inactivation

demonstrated

Although

PDH.

of fatty

inactivation

of

acid and under incu-

change in the oxidation-reduction

these authors

of PDH involved

(2),

(25) were unable to

a significant

upon the addition

in which a significant

could be expected.

mediated

on the enzymes regulating

of

in the kinase-phosphatase

However, Linn -et al.

Wieland and von Jagow-Westermann (24) and Hucho -et al. demonstrate

inhibitors

suggested that

an oxidation-reduction

535

the fatty

effect,

acid

they were

Vol. 66, No. 2, 1975

BIOCHEMICAL

unable to distinguish mitochondrial

between effects

acyl-CoA/CoASH ratio

the ATP/ADP ratiowere their

AND BIOPHYSICAL

of the NADH/NAD+ratio

on the inactivation

deemed unimportant

as this

and the intra-

of PDH. Effects

ratio

of

remained unchanged in

experiments. Utilizing

mitochondrial

in the intramitochondrial succinate

incubation

of fatty

chondrial

levels

of various

changes in PDH activity

of the PDH, without

nucleotides

enzymes.

the NADH/NAD+ratio

tion between possible

Upon inclusion

effects

of acetoacetate

that

an effect

is in agree-

the inactivation

of the NADH/NAI+ ratio

increase

level

a distinc-

or the

medium there

occurred

of PDH was likely

to that

a

not

on the PDH kinase as the ratio

in this

of

which would be consistent

experiment

the acetyl-CoA

level

and there was no change in the acetyl-CoA/

there was a significant of. acetoacetate

between the oxidation

in

of PDH could not be made.

This activation

opposite

of

of the ATP/ADP ratio

caused a 2-fold

in the incubation

of the PDH. Also,

Hence, upon addition

activation

This finding

changes in both of these ratios,

on the inactivation

remained at an undetectable

inactivation

in the acetyl-CoA/CoASH ratio.

of the adenine nucleotides

while

caused a h-fold

of octanoate

in the PDH activity.

with activation

correlation

did not involve

individual

ATP/ADP changed in a direction

CoASHratio

of these intermediates.

(14) indicating

Addition

the intramito-

in the levels

of octanoate

significant

acetyl-CoA/CoASH ratio

due to effects

Additionally,

the

in order to correlate

and a large increase

Because there occurred

increase

3 in the presence of

a change in the ATP/ADP ratio.

acid addition

on the regulatory

state

changes

were determined

with alterations the addition

which would resist

of PDHwas measured following

acetoacetate.

ment with the data of Taylor -et al. PDH upon fatty

e.g.

the activity

acid and/or

It was observed that

conditions

ATP/ADP ratio,

as the substrate,

addition

&fold

RESEARCH COMMUNICATIONS

decrease in the NADH/NAD+ratio.

to the mitochondria of mitochondrial

there was a positive

pyridine

nucleotide

and the

of the PDH.

In the presence of acetoacetate

the inclusion

536

of octanoate

in the incu-

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

k 0

537

RESEARCH COMMUNICATIONS

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

bation

in a T-fold

inactivation

medium resulted

was probably minimal

of PDH. Again,

of the ATP/ADP ratio.

change in the NADH/NAD+ratio

occurred that

not due to an effect

RESEARCH COMMUNICATIONS

upon octanoate

addition

increase

in the acetyl-CoA/CoASH ratio.

slight

increase

in NADH/NAD+had much effect

as a 2-fold

increase

the PDH activity

in NADH/NAD+discussed

the extensive

the presence of acetoacetate

inactivation

while

there

It is unlikely on the inactivation

above was necessary to convert

from 23.7 to 5.7 nmoles/min.mg

concluded that

effect

There was only a

a large

this

this

protein.

Hence, it

of PDH upon fatty

was correlated

with the large

is

acid addition increase

in

in the

acetyl-CoA/CoASH ratio. It

is interesting

to which octanoate

to compare the control

and acetoacetate

incubation

were added.

small change in the ATP/ADP ratio,

acetyl-CoA/CoASH inactivation

ratio

increased

In this

the NADH/NAD+ratio

magnitude which should have caused a significant

with the incubation case there was a decreased with a

activation

by an amount consistent

of the enzyme complex.

The resultant

of PDH, and the with an extensive

effect

on the enzyme with

these opposing changes in the NADH/NAD+and acetyl-CoA/CoASH ratios a slight

inactivation

An alternative

of the enzyme. explanation

for the effects

CoASHon the measured activity direct

effects

on the active

were the case, dilution the various

conditions

increase

described

form of PDH during

mitochondrial

That this in Table II.

incubation

and the PDH activity

the assay procedure.

suspensions

preincubated

exert If this under

the observed

was not the case, may be seen in the Although activity

Gfold

538

caused a consistent of PDH activity

remained unchanged even though the prior

of the ratios

in our experiments

dilution

of PDH, the ratios

conditions

sample was diluted

The observed correlation

NADH and acetyl-CoA

to assay should have minimized

in the measured specific

under the different

of NADH/NAD+and acetyl-CoA/

of PDH might be that

of the mitochondrial prior

changes in PDH activity. experiment

was only

to assay. of NADH/NAD+and acetyl-CoA/CoASH

suggests that

the fatty

acid mediated

Vol. 66, No. 2, 1975

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0

G. ci

4

539

F4

u

a

Vol. 66, No. 2, 1975

inactivation

BIOCHEMICAL

of PDH involves

of this

suggestion,

kinase

is stimulated

Pettit

AND BIOPHYSICAL

changes in both of the above ratios. et al. --

(26) have recently

by NADHand acetyl-CoA

CoASH. The PDH phosphatase

RESEARCH COMMUNICATIONS

is inhibited

found that

and is inhibited

by NADHand this

Supportive the PDH

by NAD+ and effect

is reversed

by NAD+. REFERENCES Linn, T. C., Pettit, F. H., and Reed, L. J. (1969) Proc. Nat. Acad. Sci. (USA) 62, 234-241. 2. Linn, T. C., Pettit, F. H., Hucho, F., and Reed, L. J. (1969) Proc. Nat. Acad. Sci. (USA) 2, 227-234. F. H., Hucho, F., Randall, D. D., and 3. Linn, T. C., Pelley, J. W., Pettit, Reed, L. J. (1972) Arch. Biochem. Biophys. s, 327-342. 4. Portenhauser, R., and Wieland, 0. (1972) Eur. J. Biochem. & 308-314. 5. Martin, B. R., Denton, R. M., Pask, H. T., and Randle, P. J. (1972) Biochem. J. 3, 763-773. 6. Wazajtys, E. I., Gottesman, D. P., and Williamson, J. R. (1974) J. Biol. Chem. 9, 1857-1865. F. H., Roche, T. E., and Reed, L. J. (1972) Biochem. Biophys. Res. 7. Pettit, Commun. 49, 563-571. a. Schuster, S. M., and Olson, M. S. (1974) J. Biol. Chem. 9, 7159-7165. 9. Roche, T. E., and Reed, L. J. (1974) Biochem. Biophys. Res. Conxnun. 59, 1341-1348. 10. Wieland, O., von Funcke, H., and Lbffler, G. (1971) Fed. Eur. Biochem. Sot. Lett. 2, 295-298. 11. Patzelt, C., LBffler, G., and Wieland, 0. H. (1973) Eur. J. Biochem. 3, 117-122. l-2. Taylor, S. I., Mukherjee, C., and Jungas, R. L. (1973) J. Biol. Chem. 248,

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Wieland, 0. H., and Portenhauser, R. (1974) Eur. J. Biochem. 3, 577-588. Taylor, S. I., Mukherjee, C., and Jungas, R. L. (1975) J. Biol. Chem. 9,

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Schneider, W. C., and Hogeboom, G. H. (1950) J. Biol. Chem. 183, 123-128. Layne, E. (1957) Methods in Enzymol. 3, 447-454. Williamson, J. R., and Corkey, B. E. 71969) Methods in Enzymol. I& 434-513. Tubbs, P. K., and Garland, P. B. (1969) Methods in Enzymol. 13, 535-551. Chem. Sanadi, D. R., Littlefield, J. W., and Bock, R. M. (1952) J.?%ol.

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Hall, L. M. (1962) Anal. Biochem. 3, 75-80. Garland, P. B., and Randle, P. J. 71964) Biochem. J. s, 6c-7~. Bremer, J. (1969) Eur. J. Biochem. g, 535-540. Wieland, O., von Jagow-Westermann, B., and Stukowski, B. (1969) HoppeSeyler's Z. Physiol. Chem. 350, 329-334. Wieland, O., and von Jagow-Westermann, B. (1969) Fed. Eur. Biochem. Sot. Letters 3, 271-274. Hucho, F., Randall, D. D., Roche, T. E., Burgett, M. W., Pelley, J. W., and Reed. L. J. (1972) Arch. Biochem. Biophys. I& 328-340. Pet-tit, F. H., Pelley, J. W., and Reed, L. J. (1975) Biochem. Biophys. Res. Commun., in the press.

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