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Biochimica et Biophysica Acta, 4 4 4 ( 1 9 7 6 ) 3 3 - - 4 2 © Elsevier Scientific Publishing Company, Amsterdam

--Printed

in T h e N e t h e r l a n d s

BBA 27980

REGULATION OF THE PYRUVATE DEHYDROGENASE ACTIVITY IN THE ISOLATED PERFUSED HEART OF GUINEA-PIGS *

HANS REINAUER

a,* a n d E R N S T R . M U L L E R - R U C H H O L T Z

b

a Biochemische A bteilung des Diabetes-Forschungsinstituts and b Institut fiir Physiologie I der Universita't Diisseldorf, Diisseldorf (G.F.R.) (Received February 16th, 1976}

Summary The activity and the interconversion of the pyruvate dehydrogenase complex has been measured in the isolated perfused working hearts of guinea-pigs. 1. The pyruvate dehydrogenase complex is transferred into the active form by high work, in anoxia, with 2,4-dinitrophenol and by perfusion without substrate. The rate of interconversion is faster in the perfused heart than in the homogenate. 2. The active form of the pyruvate dehydrogenase complex limits the pyruvate oxidation. There is a close correlation between the pyruvate utilization in the perfused hearts and the pyruvate dehydrogenase of the active form in the homogenates of the same hearts. 3. The "adenyla~e energy charge" of the cells is considered as the main regulating factor of the interconversion of the pyruvate dehydrogenase complex as seen in experiments with anoxia, dinitrophenol and high work. The inactivation of the pyruvate dehydrogenase complex by acetyl CoA can be overcome by decreasing ATP/ADP ratios.

Introduction It is well established that in the heart muscle the activity of the pyruvate dehydrogenase complex is regulated by interconversion of the pyruvate decarboxylase, which is the first and rate limiting enzyme of the multienzyme complex [1--4]. The regulation by interconversion of the pyruvate dehydrogenase complex * Parts of this paper have been presented at the 8th Kongress der Deutschen Diabetes Gesellschaft, M/inehen 1 9 7 3 . * C o r r e s p o n d e n c e to: Prof. Dr. H. Reinauer, Diabetes-Forschungsinstitut, A u f ' m H e n n e k a m p 65, 4 Dfisseldorf 1, G.F.R.

34 is without any metabolic relevance if the active form of the pyruvate dehydrogenase complex does not limit the pyruvate utilization. In earlier investigations we could show that in heart muscle there is a close quantitative correlation between pyruvate decarboxylation and oxygen consumption [ 5]. The aim of the present paper was to show the regulating effect of heart work and of substrates on the interconversion of the pyruvate dehydrogenase complex. The extensive perfusion m e t h o d has been used to guarantee steadystate conditions in heart performance and metabolism. Materials D-[UJ4C]Glucose (3 Ci/mol), [1J4C]pyruvic acid, sodium sa]t (12 Ci/mol), [2-14C]pyruvic acid, sodium salt (10 Ci/mol), [UJ4C]acetic acid, sodium salt (59 Ci/mol) and L-[U-'4C]malic acid (50 Ci/mol) were from Amersham Buchler GmbH, Braunschweig. All chemicals were of analytical grade. The enzymes for the analysis came from Boehringer, Mannheim. Insulin was obtained from Hoechst AG.

Animals The hearts of male guinea-pigs (500--700 g) were used for the perfusion experiments. The animals were maintained on standard laboratory chow (Sniff) ad libitum. The food was withdrawn 14 h before the experiments. 30 min before the experiments heparin (500 units/kg) was injected intreperitoneally. The animals were anaesthetized by injections of urethan (1.5 g/kg} intraperitoneally. Perfusion apparatus Two perfusion systems were used. 1. Perfusion system according to Langendorff [6] where hearts are perfused through a cannula inserted into the aorta [ 5]. 2. The working heart system, which enabled the inflow volume into the left atrium and the perfusion pressure to be varied by altering the aortic pressure (Fig. 1; ref. 7). In both systems there was no recirculation of the perfusion medium, which consisted of bicarbonate buffered salt solution [8] gassed with O2/CO2 (95 : 5). The perfusion medium contained no albumin and was maintained at 37°C. If not indicated otherwise the following concentrations of substrates and effectors were used: 10 mM glucose, 5 mM pyruvate, 0.1 mM 2,4-dinitrophenol, 5 IU/1 insulin. Procedure For technical reason the perfusion experiments began with the first system (flow rate 15 ml/min). After 30 min steady-state conditions were reached and in the following 20 min the experiments were performed. High work conditions were obtained by changing the Langendorff system to the atrial system (Fig. 1 ). The perfusion medium now passed through the left atrium into the left ventricle, and from here into the aorta and the coronary vessels (inflow 20 ml/min, aortic pressure 60 mm Hg). In studying the effect of increased cardiac work,

35

LANGENDORFF INFLOW

ATRIAL INFLOW INFLOW RESISTANCE

•

, 1-METER /

AO T,C

r

OUTFLOW

II 4"

RESISTANCE

~

"'~ [

~

II

I

I

II

I

I I:1 J:l FLOWMETER

I . I "TT

J In1,, o II ,

~

u, ~

//_

E~,~, ~ l ~%,e.,~

STATHAM

!I:iM:EL;Cy k /~J,.~ If'

'

S,.THAM FLOWMETER"

Fig. 1. P e r f u s i o n s y s t e m for the isolated h e a r t . I n this s y s t e m the h e a r t c a n be perfused according to Lang e n d o r f f [ 6 ] a n d a l t e r n a t i v e l y u n d e r l o w a n d high w o r k c o n d i t i o n s . High w o r k c a n b e a c h i e v e d b y increasing t h e i n f l o w v o l u m e i n t o t h e left a t r i u m a n d t h e a o r t i c o u t f l o w r e s i s t a n c e ( f o r details see M e t h o d s a n d ref. 7).

the inflow volume and the aortic pressure were changed to 40 ml/min and 100 mm Hg, respectively. The whole perfusion period lasted for 45 min. The following parameters were monitored continuously by means of the 8 channel Beckmann R dynograph: left ventricular pressure, diastolic pressure, the rate of change of left intraventricular pressure (dp/dt), the aortic pressure, the coronary flow and the pO2 in the arterial and venous system. By this way, the oxygen consumption and the performance of the isolated heart could be monitored and steady-state conditions could be guaranteed.

Analytical methods The utilization of [U-14C]glucose, [U-14C]acetate, [2J4C]pyruvate and [U14C] malate in the heart could be measured by trapping 14CO2 in the outflowing (coronary venous) medium. For this purpose aliquot of the "venous" perfusion medium was collected, filled in a vessel according to Widmark, acidified, the 14CO2 absorbed in Hyamine® and the counts measured in a liquid scintillation counter. In the experiments with [~4C]acetate there was a high absorption of [J4C]acetic acid together with 14CO2 in Hyamine after acidification of the perfusion medium. Therefore, the liberated 14CO2 in the "venous" perfusion medium has been precipitated with Ba(OH)2 to separate ~4CO2 from [~4C]acetate. At the end of the perfusion experiments, the isolated hearts were immediately frozen with a Wollenberger clamp, precooled in liquid nitrogen. In the frozen heart tissue, the activity of the pyruvate dehydrogenase complex [4] and its interconversion state were measured [2,3]. Another part of the frozen tissue

36

was pulverized on a mortar while cooling with liquid nitrogen and extracted with HClO4. In the neutralized extract ATP [ 9 ] , ADP and AMP [ 1 0 ] , creatine [ 1 1 ] , creatine phosphate [ 1 2 ] , lactate and pyruvate [ 13] were estimated. A separate sample of frozen heart powder was analyzed for glycogen [ 14].

Calculations Substrate and c o e n z y m e contents are expressed in pmol or nmol per g wet wt. The o x y g e n consumption was measured as gl 02 • min • g wet wt. or calculated as pmol 02 • min • g wet wt. To avoid the effect of edema in the hearts the values were referred to dry weight or corrected on the basis of protein content per wet weight of in vivo hearts. The data are given as means -+ standard error (~ -+ S.E.). Results The rate of interconversion of the pyruvate dehydrogenase complex is faster in the isolated perfused heart than in the in vitro incubations of the homogenate, where w i t h o u t addition of pyruvate dehydrogenase phosphatase 3 0 - - 4 0 min are necessary to fully activate this complex [ 1 , 2 ] . During the perfusion of the isolated heart with 5 mM pyruvate, the active state of pyruvate dehydrogenase complex is as high as 57% of the total activity (Fig. 2). If the heart contractions are suppressed by addition of KC1 (final concentration 26.85 mM) to the perfusion medium, the o x y g e n consumption decreases to 26 pl 02 per min, whereas the activity of the active form does n o t change significantly (54%). After addition of 0.1 mM 2,4-dinitrophenol to the perfusion medium,

pyruvate ~'o z )41021min

pyruvate-KC[

p y r u v a t e - K Q - 2,4 - D N P

% PDH a

140 -

120 -

/

100 -

80-

60-

40-

20-

0 20

30 n=6

40

n=5

Fig. 2. O x y g e n c o n s u m p t i o n of t h e i s o l a t e d p e r f u s e d heart o f guinea-Pig. Substrate: p y r u v a t e 5 m M . T h e h e a r t p e r f o r m a n c e was s u p p r e s s e d b y 2 6 . 8 5 mM KC1, t h e r e a f t e r , the o x y d a t i v e p h o s p h o r y l a t i o n w a s unc o u p l e d b y 0.1 m M 2 , 4 - d i n i t r o p h e n o l . T h e bars r e p r e s e n t t h e activity o f the active f o r m in per c e n t o f the t o t a l a c t i v i t y . PDHa, active f o r m o f p y r u v a t e d e h y d r o g e n a s e .

37 the oxygen consumption and the activity of the active form of the pyruvate dehydrogenase complex increase within 1 minute (Fig. 2). By comparing the activity of the active form with the oxygen consumption and with the oxidation of glucose and pyruvate in the perfused heart of fasted animals, close correlations between these parameters are found (Table I). With pyruvate as substrate the following correlations between the active form (%) and Vo2 (pl O: per min) were found: r = 0.881 + 0.178, p < 0.001; y = 24.25 + 0.403 x. In the fed animals the corresponding values are r = 0.483 -+ 0.242, p < 0.05; y = 57.23 + 0.181 x. In the fed animals the activity of the active form in the perfused heart is usually higher than the activity which has been calculated from the generation of ~4CO2 or from the oxygen consumption (see ref. 7). On the other hand there is a significant negative correlation between the active state of pyruvate dehydrogenase complex and the ATP content in the heart muscle under several experimental conditions (Tables II and III). The changes in the content of energy-rich compounds and in the activity of the active form are better illustrated in the experiments with anoxia. In anoxia the activity of the active form increases while the ATP/ADP and creatine phosphate/creatine ratios diminish significantly. The increased heart work achieved by changing the inflow to the left atrium from 20 to 40 ml/min and the aortic pressure from 60 mm Hg to 100 mm Hg is followed by an increase of oxygen consumption from 309 ± 20 pl O: • min • g to 605 ± 36 pl O : . min • g. With glucose as substrate the activity of the active form increases significantly in the hearts of fed and fasted animals (Fig. 3). In the corresponding experiments with acetate as substrate, high work increases oxygen consumption but not the activity of the active form of pyruvate dehydrogenase. Nutritional state had a minor effect under these experimental conditions. From these experiments it becomes evident that acetate or a metabolite of acetate suppresses the activation of the pyruvate dehydrogenase complex by TABLE I C O M P A R I S O N OF A C T I V I T Y OF T H E A C T I V E F O R M OF P Y R U V A T E D E H Y D R O G E N A S E , O X Y G E N C O N S U M P T I O N A N D P R O D U C T I O N OF T H E 14CO2 F R O M [ U - 1 4 C ] G L U C O S E A N D [ 2 - 1 4 C ] P Y R U V A T E IN T H E I I E A R T M U S C L E O F F A S T E D A N I M A L S i?pyr, p y r u v a t e u t i l i z a t i o n as m e a s u r e d b y t h e l i b e r a t i o n of 14CO2 f r o m [ 2 - 1 4 C ] p y r u v a t e . F o r c o m p a r i s o n of the p y r u v a t e o x i d a t i o n w i t h 1702 (= o x y g e n c o n s u m p t i o n ) t h e v a l u e s of 1702 h a v e to be d i v i d e d b y 2.5. Initial c o n c e n t r a t i o n s o f g l u c o s e and p y r u v a t e w e r e 5 and 10 m M r e s p e c t i v e l y .

Glucose

VO2 ( / ~ m o l / m i n p e r g)

A c t i v e f o r m of pyruvate dehydrogcnase ( p m o l / m i n p e r g)

I/Pyr. f r o m 14CO2 ( p m o l / m i n per g)

3.97 + 2 . 2 3

1.07 + 0.26

1.07 +- 0 . 0 2

6.21 + 0 . 5 4

2.18 + 0.29

1.35 -+ 0 . 0 7

5.80 ± 1.78

1.84 + 0 . 1 2

1.94 ± 0 . 4 0

8.61 ± 2.41

2.74 ± 0 . 7 6

2.76 * 0.39

n=4

Glucose + DNP n=5

Pyruvate n=5 Pyruvate + DNP n=ll

38 T A B L E II CORRELATIONS BETWEEN ACTIVITY OF THE ACTIVE FORM OF PYRUVATE DEHYDROGENASE (PDHa) AND ATP CONTENT, ATP/ADP RATIO, "ENERGY CHARGE" AND CREATINE PHOSP H A T E C O N T E N T IN T H E I S O L A T E D P E R F U S E D G U I N E A - P I G t t E A R T C a l c u l a t i o n s o f c o r r e l a t i o n c o e f f i c i e n t (r) a n d r e g r e s s i o n l i n e are p e r f o r m e d a c c o r d i n g t o r e f s . 3 3 a n d 3 4 w i t h t h e d a t a o f T a b l e I I I ( a c e t a t e ± a n o x i a ) . S u b s t r a t e : a c e t i c a c i d , s o d i u m salt. x

y

.~ = a y x + b y x • x

r ÷ sr *

p

PDH a PDH a PDH a

ATP ATP/ADP A T P + 1/2 A D P

y = 6.727 -- 1.162 x y = 8.524 -- 1.424 x

--0.931 ~ 0.110 ---0.920 * 0 . 1 1 8