Hutson, Nancy J.

Randle, Philip J.

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Volume 92, number 1 FEBS LETTERS August 1978 
ENHANCED ACTIVITY OF PYRUVATE DEHYDROGENASE KINASE IN RAT HEART 
MITOCHONDRIA IN ALLOXAN-DIABETES OR STARVATION 
Nancy J. HUTSON and Philip J. RANDLE 
Nuffield Department of Clinical Biochemistry, Radcliffe Infirmary, Oxford, OX2 6H.K England 
Received 8 June 1978 
1. Introduction 
The PDH complex of animal tissues is converted 
to PDHP complex and inactivated by an intrinsic 
PDH kinase (utilizing MgATP*-) and reactivated by 
PDHP phosphatase [l] . The kinase and phosphatase 
reactions constitute a cycle with the proportions of 
PDH and PDHP complexes depending on their relative 
rates. Alloxan-diabetes or starvation in the rat mark- 
edly decrease the concentration of PDH complex 
without changing the total concentration (PDH + 
PDHP) [2] . The mechanism by which this is brought 
about is not completely understood. The PDH kinase 
reaction is activated by increasing concentration 
ratios of ATP/ADP, NADH/NAD’ and acetyl CoA/ 
CoA and inhibited by pyruvate [ 1,3,4] . However, 
changes in the concentrations of these effecters do 
not explain the decreased proportion of PDH com- 
plex in heart mitochondria in diabetes [ 5,6] . Reacti- 
vation of PDH complex by PDHP phosphatase is 
inhibited in heart mitochondria of starved or diabetic 
rats [7] . This inhibition may be due to the phos- 
phorylation of sites additional to an inactivating site 
in the PDH complex [7,8] and is therefore dependent 
on the PDH kinase reaction. Evidence i: given here 
that the PDH kinase reaction is accelerated in heart 
mitochondria from alloxan-diabetic or 48 h starved 
rats by a novel mechanism which does not appear to 
depend on known metabolite effecters of PDH kinase. 
Abbreviations: DTT, dithiothreitol; EGTA, ethanedioxybis 
(ethylamine)-tetra-acetate; PDH, pyruvate dehydrogenase; 
PDHP, pyruvate dehydrogenase phosphate. PDH complex is 
(EC 1.2.4.1 + EC 2.3.1.12 + EC 1.6.4.3); ATPase, adenosine 
triphosphatase; SDS, sodium dodecyl sulphate 
ElsevierfNorth-Holland Biomedical Press 
2. Experimental 
2.1. Materials 
Nagarse was from Hughes and Hughes Ltd., 
Romford, Essex. Sources of all other reagents and of 
animals, and details of induction of alloxan-diabetes 
are given in references [5-71. Pig heart [32P] PDHP 
complex (1.7 nmol P/unit PDH complex) was prepared 
as in [8] . One unit of enzyme activity is 1 pmol 
substrate utilized or product formed/min at 30°C. 
2.2. Methods 
PDH complex in mitochondrial extracts was assayed 
spectrophotometrically by coupling to arylamine 
acetyltransferase [5] . ATPase was assayed by release 
of 32Pi from [T-~~P]ATP [9] . Details of SDS poly- 
acrylamide disc gel electrophoreses are given in [lo] . 
Rat heart mitochondria were prepared with the 
bacterial protease Nagarse [ 1 l] because this method 
gave minimal contamination with oligomycin-insensi- 
tive ATPase. Mitochondria (1.1 mg protein) were 
incubated for 10 min at 30°C in KCl medium without 
respiratory substrate (to convert PDHP complex into 
PDH complex [ 51) separated by centrifugation and 
frozen [5] . The pellets were extracted by freezing 
in liquid N2 and thawing at 30°C (X 3) in 30 mM 
potassium phosphate/ 10 mM EGTA/ 1 mM Niu-p- 
tosyl-L-lysine chloromethyl ketone/S mM DTT/ 
25 fig/ml oligomycin/pH 7.0 (2.75 mg mitochondrial 
protein/ml). PDH kinase was assayed in these extracts 
by the decline in activity of PDH complex and by 
formation of 32P-protein [ 121 . After assay of zero 
time PDH complex, extracts (600 4) were pre- 
incubated for 2 min at 30°C and the PDH kinase 
73 
Volume 92. number 1 FEBS LETTERS August 1978 
reaction initiated with 18 1.11 of 10 mM [r-32P]ATP 
(45 pCi/pmol). Samples were taken for assay at times 
shown in the figures. 
Magnesium required for the kinase reaction (as 
MgATP’-) was derived solely from the mitochondria 
and concentrations (in ng-atoms Mg/ml of extract) 
were 90 f 12 (control) and 80 f 5.3 (diabetic) 
(mean + SEM for at least six determinations by atomic 
absorption spectrometry). The computed concentra- 
tion of MgATP*- [see 91 was approximately 50 PM. 
It was necessary to work at low concentrations of 
Mg*+ (computed as 5 PM) to minimise activities of 
PDHP phosphatase (which requires Mg*‘) and ATPase. 
With these conditions, PDHP phosphatase was totally 
inactive; there was no detectable loss of protein-bound- 
32P on incubation of 0.2 unit [ 32P]PDHP with 200 ~1 
of mitochondrial extract for 9 min at 30°C. ATPase 
removed l-IS% of ATP/min. Under these conditions 
there were no significant differences in PDHP phos- 
phatase or ATPase activities between extracts from 
control, diabetic or starved animals. Extracts incubated 
without ATP showed no more than 10% loss of PDH 
activity over 6 min. 
3. Results and discussion 
The PDH kinase activity was significantly enhanced 
(relative to controls) in extracts of heart mitochondria 
from alloxan-diabetic or 48 h starved rats. This was 
apparent in the more rapid initial rates of inactivation 
of PDH complex (fig.1, panels a, b) and of incorpo- 
ration of 32P into protein (fig.1, panels c, d). With all 
extracts, inactivation of PDH complex was essentially 
complete in 6 min (fig.1) whereas 32P incorporation 
continued beyond this time (not shown). These 
properties of the PDH kinase reaction in mitochondrial 
extracts are very similar to those observed with 
purified pig heart PDH complex [lo] SDS polyacryl- 
amide disc gel electrophoresis of extracts (control and 
diabetic) after 30 min of incubation with [Y-~*P] ATP 
showed only a single band of radioactivity with the 
same mobility as the 32P-labelled o-subunit ofpuritied 
pig heart [ 32P]PDH complex (not shown). This is 
consistent with other evidence that this is the only 
major peptide in which phosphorylation can be 
detected in isolated rat heart mitochondria [7,13] . 
The PDH kinase reaction is activated by NADH 
74 
d 
Fig.1. PDH kinase activity in extracts of heart mitochondria 
from control (o), alloxandiabetrc (A), and 48 h starved (m) 
rats. Extracts were prepared as described in experimental 
section and pre-incubated at 30°C for 2 mm. The PDH kinase 
reaction was initiated by adding [T-~‘P]ATP to 0.3 mM. 
Imtral PDH complex activities (mean + SEM in mU/mg 
protein) were. in panels (a) and (c) 101 r 7.6 (control), 
88 + 7.3 (diabetic); in panels (b) and (d) 109 t 3.9 (control), 
106 + 4.6 (starved); means based on not less than 4 observa- 
tions. Points shown as mean ? SEM. For differences (control 
vs. diabetic) or (control vs. starved) *P<O.Ol; +P<O.O5. 
and acetyl CoA and inhibited by NAD’, CoA and 
ADP [ 1,361 . It was important to investigate the 
possibility that the difference in kinase activities 
between extracts of control and diabetic mitochondria 
might be due to differences in the concentration ratios 
of ATP/ADP, NADH/NAD’ and acetyl CoA/CoA. 
The mitochondrial contents of ATP and ADP [6] are 
less than 2% of the ATP added to initiate the kinase 
reaction in the present study and, moreover, no 
difference of significance between control and diabetic 
mitochondria was observed [6]. The mitochondrial 
contents of NADH and acetyl CoA after no substrate 
incubation are too low for reliable assay even by 
sensitive methods. Such estimates as are available [6] 
suggest hat the concentration ratios in the present 
Volume 92, number 1 FEBS LETTERS August 1978 
kinase assays would be no greater than 0.15 PM 
NADH/lO PM NAD’and 0.36 nM acetyl CoA/3 22 PM 
CoA. The actual ratios are likely to be much lower as 
mitochondrial extracts oxidise NADH and hydrolyse 
acetyl CoA. The rates of the kinase reaction in control 
and diabetic mitochondria were not altered by addi- 
tion of CoA (to 100 PM added CoA) plus NAD’ 
(to 800 MM added NAD’), or of oxaloacetate (to 
100 PM) during preincubation and incubation. Activa- 
tion of the bovine kidney and pig heart PDH kinase 
reaction by NADH or acetyl CoA is reversed by NAD’ 
or CoA [3-51. The mitochondrial extracts in the 
kinase assays in this paper contained citrate synthase 
(3 U/ml) and malate dehydrogenase (8 U/ml). Addi- 
tion of oxaloacetate should therefore lead to removal 
of acetyl CoA (as citrate) and of NADH (by oxida- 
tion). It seems unlikely therefore that the enhanced 
kinase activity in extracts of heart mitochondria 
from diabetic (or starved) rats is due to the action 
of known metabolite effecters of the kinase. 
The PDH kinase reaction is inhibited by pyruvate 
[ 1,9] and pyruvate increases the proportion of PDH 
complex in heart mitochondria [5-71 . The effect of 
pyruvate in heart mitochondria is markedly reduced 
by alloxandiabetes or starvation [S-7] . Pyruvate 
(2.7 mM) inhibited the PDH kinase reaction in extracts 
of rat heart mitochondria (compare panels bearing 
the same letter code in figs.1 and 2). The enhanced 
activity of the kinase reaction (relative to controls) in 
extracts of mitochondria from diabetic or starved rats 
was retained in the presence of 2.7 mM pyruvate 
(fig.2, panels a-d). 
The mechanism of the enhanced PDH kinase 
activity in heart mitochondria in diabetes and starva- 
tion shown in these studies is not known. The mecha- 
nism must involve rather stable factor(s) because it 
persists through isolation, incubation and extraction 
of mitochondria. Possibilities include increased con- 
centration of PDH kinase in the PDH complex or 
activation of the kinase reaction by covalent modifica- 
tion of the complex. Acetylation or reduction of 
lipoyl residues in the complex, which have been 
reported to activate the PDH kinase reaction [ 141 , 
are unlikely to be involved here as the kinase activa- 
tion observed in this study was not reversed by addi- 
tion of CoA + NAD’. The significance of this activa- 
tion of the kinase in diabetes and starvation may be 
twofold. It may facilitate inactivation of the complex 
c d 
t I 
0 2 4 6 0 2 4 6 
Fig.2. PDH kinase activities in extracts of heart mitochon- 
dria from control (o), alloxandiabetic (A), and 48 h starved 
(m) rats. Details of incubation as in fig.1 except that sodium 
pyruvate (2.75 mM) was present throughout pre-incubation 
and incubation. Initial PDH complex activities were, in 
panels (a) and (c) 108 ? 7.6 (control), 91 f 7.7 (diabetic); 
in panels (b) and (d) 103 r 11.3 (control), 71 ? 7.2 (starved); 
means based on not less than 4 observations. Points shown 
as mean fr SEM. For differences (control vs diabetic) or 
(control vs starved) *P<O.Ol; +P<O.OS. 
via phosphorylation of an inactivating site as shown 
in this study. It may also inhibit reactivation of PDHP 
complex by PDHP phosphatase by facilitating the 
phosphorylation of sites additional to the inactivating 
site [8] if rat heart PDH complex is similar to pig 
heart PDH complex. 
Acknowledgements 
NJH is a US Public Health Service Research Fellow. 
We thankMrs M. C. Sugden for disc gel electrophoresis, 
Mrs P. M. Radcliffe and Mr M. Lowry for technical 
assistance and Mr D. Quantrill for atomic absorption 
measurements. Supported by grants from the Medical 
Research Council and British Diabetic Association. 
75 
Volume 92, number 1 FEBS LETTERS August 1978 
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[ 1] Linn, T. C., Pettit, 1:. Il., Hucho, F. and Reed, L. J. 
(1969) Proc. Natl. Acad. Sci. USA 64,227-234. 
[2] Wieland, 0. H.. Siess, E. A., Schulze-Wethmar, F. H., 
von Funcke, 1~. and Winton. D. (1971) Arch. Biochem. 
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[3] Pettit, 1:. H., Pelley. J. W. and Reed, L J. (1975) 
Biochem. Biophys. Res. Commun. 65, 575-582. 
[4] Cooper, R. H., Randle, P. J. and Denton, R. M. (1975) 
Nature (London) 257, 8088809. 
[5] Kerbey. A. L., Randle, P. J., Cooper, R. H.. 
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[6] Kerbey, A. L., Radcliffe, P. M. and Randle, P. J. (1977) 
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[7] Hutson, N. J., Kerbey, A. L., Randle. P. J. and Sugden, 
P. H. (1978) Biochem. J. in press. 
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[lo] Sugden, P. H. and Randle, P. J. (1978) Biochem. J. 
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76 


Enhanced activity of pyruvate dehydrogenase kinase in rat heart mitochondria in alloxan-diabetes or starvation 

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Enhanced activity of pyruvate dehydrogenase kinase in rat heart mitochondria in alloxan-diabetes or starvation

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