BIOCHEMICAL
Vol. 83, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
Pages
August 29,1978
1422-1429
CRABTREE EFFECT INDUCED BY FRUCTOSE IN ISOLATED HEPATOCYTES FROM FED RATS E. Chico,
J.S.
Olavarrla
and I.
Ndfiez
de Castro
Centro
de Biologfa Molecular, C.S.I.C., Facultad de Universidad Autanoma, Cantoblanco, Madrid Ciencias,
(34),
and G. GimGnez-Gallego Departamento Universidad Received
de Fisiologfa AutBnoma,
July
Vegetal,
Facultad
Cantoblanco,
Madrid
de Ciencias, (34)
7,1978
Summary.In hepatocytes isolated from fed rats, the addition of fructose caused an inhibition of respiration. In hepatocytes isolated from starved rats the Crabtree effect was not observed. No difference in oxygen uptake was found by addition of glucose to hepatocytes from fed or starved animals. The inhibition of respiration was parallel with a rise in the glycolytic flux and the oxidation of the mitochondrial respiratory carriers. The metabolic conditions in which the Crabtree effect can be operative in liver cells are discussed. The inhibition and the high aerobic
of respiration glycolysis
energetic
of
first (2).
mechanism
observed Hassinen
tumor
in a normal and Ylikahri
by glucose (Crabtree effect) have been considered as a common
cells
(1).
The Crabtree
effect
tissue in the retina by Cohen (3) have reported an inhibition
was and Noel1 of
respiration induced by fructose in perfused rat liver. This menon appears simultaneously with changes in the cytochromes dox state.
Therefore,
there is a mutual as was previously
regulation suggested
phenore-
between glycofor ascites
lysis and respiration, tumor cells (4). On the contrary, Seglen (5), using parenchemal cells isolated from the livers of 16 hour fasted rats, claimed a lack of the Crabtree effect in hepatocytes incubated with 20 mM fructose. In the present report the Crabtree effect caused by fructose in hepatocytes isolated from rat liver is further investigated, in order to ascertain the metabolic circumstances in which the inhibition of respiration induced by fructose takes place. 0006-291X/78/0834-1422$01.00/0 Copyright All rights
0 1978 by Academic Press, Inc. of reproduction in any form reserved.
1422
BIOCHEMICAL
Vol. 83, No. 4, 1978
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
MATERIAL AND METHODS D-glucose and D-fructose were from E. Merck, Darmstadt. NAD+, NADH, AT!?, N-acetyl D-glucosamine, D-glyceraldehyde, Ficoll and lactate dehydrogenase were from Sigma Chemical comBovine serum albumin was purchased by Metrix-Armour Pharpaw. maceutical Company, Chicago. All other enzymes used for the isolation of hepatocytes and for the determination of metabolites and enzymes activities were obtained from Boehringer Manhein GmbH. Female Wistar rats weighing 150-200 g were used for liver cells preparation. The hepatocytes were isolated by the method of Berry and Friend (6) modkfied by Cornell et al. (7). The isolated liver cellsr4 - 5 x 10 cells per ml, were incubated at 37°C in a Grant (SS 30) metabolic shaker at 140 strokes/min in 50 ml glass flasks under an atmosphere of 95% O2 and 5% CO . The incubation medium was 8.25 mM phosphate buffer pH 7.4, O,l? M NaCl, 4.6 mM KC1 and 2.5% (w/v) bovine serum albumin dialyzed overnight. After 60 min, 0.2 ml of 60% (w/v) HC104 were added and the mixture was centrifuged. The supernatant was neutralized with 40% (w/v) KOH and the potassium perchlorate removed by centrifugation. The neutralized supernatant was used for determination of lactic acid. Oxygen uptake was measured with a Oxygraph K-1C (Gilson Medical Electronics, Inc) equipped with a thermostated 2 ml cuvette and with a continuous recording system. 100 1 of glucose or fructose were added to obtain a final volume o P 2 ml at desired concentration. Addition of an equivalent amount of buffer does not affect the cell respiration. The initial concentration of 02 in the buffer was assumed to be 0.25 pmoles/ml. Difference spectra of cytochromes were recorded at room temperature using a Perkin-Elmer Hitachi Model 356 spectrophotometer equipped with a Tracer-Northen Digital Signal Averager. The cells were suspended in the incubation buffer added with 10% Ficoll. The cytochrome contents were calculated according to the methods of Wanneste (8). RESULTS AND DISCUSSION
tion
Chance and Hess (9) have reported that of glycolysis occurs when the respiration
glucose in ascites tumor (Crabtree effect).observed when fructose
was added
a parallel stimulais inhibited by
cells. The inhibition of in isolated hepatocytes to
a suspension
of
cells
respiration from fed respiring
rats endo-
genous substrates was parallel with a rise in the glycolytic flux measured by the lactic acid production. As is shown in Table 1, 10 mM fructose seemed to be the best concentration in producing such as inhibition. The smaller inhibition of respiration caused by 20 mM fructose could be explained since the fructose load in liver causes an increase of fructose-l-P and IMP, and according to Woods et al. (10) this nucleotide inhibits the
1423
+ lb L\3 A
mM) mM)
(10 mM) 5 mM)
3.720.3 4.7iO.2
9.1f0.7 9.120.7 4.620.2
(5) (3)
(5) (4) (4)
Crabtree
effect
415238 360217
902 6 188217 363220
lactate formation (nmoles/mg dry weight/h)
on the
1
induced
** ** **
12.021.4 **
in
113215 145235 210+10 (3)
462 6 71210 (3) (4)
(5) (3)
hepatocytes.
lactate formation (nmoles/mg dry weight/h)
rats
isolated
Starved 02 uptake (ngram atom/min /mg dry weight)
by fructose
* Cells respiring endogenous substrates. ** No change in oxygen uptake was obtained in hepatocytes isolated from 48 h starved rats when glucose or fructose were added. The results are means of replicate experiments + S.E.M. The numbers of experiments are indicated in parenthesis.
fructose(l0 fructose(20
glucose fructose(
*
Fed rats
state
02 uptake (ngram atom/min /mg dry weight)
of nutrition
Final hexose concentration
Influence
Table
Vol. 83, No. 4, 1978
reaction
BIOCHEMICAL
catalyzedby
aldolase
48 hour starved rats the concentration of fructose. hepatocytes in agreement 11)
rats
fed
liver. isolated
period
ad libitum
(16 hours).
of hepatocytes A transient
in
when
the
in
these
fructose
was added
as Chance activation
fructokinase
There is uptake (ngram
their experiments with perthe results and discussion of
hepatocytes
for
the rate of The redox
was added
results
we have
to hepatocytes
reaction
with
(r= weight)
and support
common substrates
to
a steady fructose
electrons change in
transport. cytochromes
resulting from the from fed rats is
there
of
in fed rats. respiration
previous the
These and
hypothesis Crabtree
that the is due to
on transition
addition shown in
oxygen of lac-
to
of
effect
inhibition a decrease the
new
of fructose to hepatoFig. 1. When fructose
-a3 (59%), cyto chrome -c (32%) and cytochrome the cells -b (40%) as compared with respiring endogenous substrates. The spectra recorded 5 and 10 min after the addition of fructose did not show significative variation.Hassinen of cytochromes intact tissue
was an oxidation
decrease
state was obtaiwas exhausted.
weight) between explain
long) endoge-
0.998) between the and the reciprocal
the
found
in ascites tuATP consump-
a simultaneous
2 minutes, until the
the
in oxysuspension
respiring
and Hess (12) observed is probably due to the
a good correlation atom/min/mg dry
in
steady state cytes isolated
the
the hepatocytes at the end of
circumstances
(13). More recently, Sauer (14) has reported of respiration in tumor cells by glycolysis in
with
because animals
tic acid production (nmoles/min/mg dry results suggest an inverse relationship competition
at any with
rats. Our results are of Hassinen et a1.(3,
hand, in contradiction
In
from
effect was not observed results were obtained
is not operative. No difference when glucose was added to the
in the ATP/ADP ratio. After ned which remained unchanged
glycolysis
isolated
isolated from starved or fed animals. activation of respiration (about 1 min
nous substrates, mor cells. This tion
RESEARCH COMMUNICATIONS
hepatocytes
could be explained were obtained from
that the Crabtree effect gen uptake was observed
occurred
the
Crabtree Similar
On the other hepatocytes
of Hassinen et al. isolated by Seglen fasting
B. In
isolated from 24 hour fasted with the earlier observation
who used
fused rat Seglen in
AND BIOPHYSICAL
cytochrome
2,
and Ylikahri (3) have also observed the oxidation a and c and a small reduction of cytochrome -b in (liver lobe in an area of 2-3 mm thick) pefused
1425
Vol. 83, No. 4, 1978
I
525 Fig.
BIOCHEMiCAL
I
I
545
565
I
565
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
I
I
L
605
625
645
nm
1 Difference spectra for aerobic suspensions of hepatocytes isolated from fed rats. A) Difference of absorbancy for cells metabolizing endogenous substrates and those oxidized with a few small crystals of potassium ferricyanide. B) Difference of absorbancy for cell suspensions
1426
Vol. 83, No. 4, 1978
8lOCHEMlCAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
with fructose. On the other hand, the addition of glucose a small reduction of cytochromes & and c. The redox state cytochrome
a remained
unchanged
as expected
of glucose to induce the Crabtree fructose was added to a suspension 24 h fasted changed.
animals
the
redox
from
state
of
the
cytochromes
fructokinase, aldolase B andtiiosekinase, the Hers pathway for fructose metabolism,
liver
from
fed
and starved
were found in the to be constitutive
activities in the
animals. of adult
No significant
these enzymes. These rat, without relation
hepatocytes
may be related avoids the regulatory
that
hexokinase sites for
diphosphate in at least twice results).
to
the
fact
isolated the
The second
control
enzymes seem to nutri-
(15) who used The Crabtree
metabolic
and phosphofructokinase glycolysis. Moreover,
fed animals (3.3 those of starved
of
differences
effect
in
was un-
the three enzymes were measured in
et al. control.
by fructose
When from
to fructose the activities
tional condition, according to Sillero high fructose diet and a standard diet induced
inability
effect in hepatocytes. of hepatocytes isolated
In order to explain the different response hepatocytes isolated from fed and starved rats, of of
the
caused of
from
fed
pathway
for
steps, levels
the of
the
first fructose
t 0.6 nmoles/mg dry weight) are animals (E. Chico, unpublished
site
at
pyruvate
Since in starved animals fructose is neogenic substrate (5) and pyruvate kinase
kinase
is
activated
starved
the
Crabtree
effect
addition
of
for animals.
a very good glucoactivity is likely
to be low (16) at physiological concentration ruvate, the glycolytic flux may be decreased Consequently,
rats
fructose
by fructose diphosphate and competes with the mitochondria ADP and consequently respiration would be inhibited in fed
ces.
a
does
of phosphoenolpyin these cirumstannot
take
place
in
rats.
5 minutes metabolizing indicated
after in
10 mM fructose
and those
endogenous substrate. C) Same conditions as B, but glucose was added instead of fructose.
Allmixturescontained 4.5 x lo6 cells/ml. The baseline was recorded 5 minutes after preparation of the suspensions, just before addition of substrate or potassium ferricyanide.
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Vol. 83, No. 4, 1978
BIOCHEMICAL
Table 2. Endogenous different pH values liver of fed rats.
respiration by fructose
PH
AND BIOPHYSICAL
and Crabtree in hepatocytes
RESEARCH COMMUNICATIONS
effect induced at isolated from
7.0
7.2
Endogenous respiration
8.420.4
8.1iO.l
8.OiO.2
8.120.6
Fructose (10 ml)
4.820.6
3.320.1
4.820.1
4.9io.2
The results
are means of
3-4 different
7.4
experiments
7.6
i
S.E.M.
The claim of Seglen, that the Crabtree effect in perfused liver is an artifact caused by acidification of the medium by fructose metabolism should be reviewed. As shown in Table 2 in isolated hepatocytes the respiration was independent of pH changes in the medium in the range of pH 7-7.6 and a clear Crabtree effect induced by fructose was observed. The results presented here confirm the existence of the Crabtree effect induced by fructose in fed rat liver. The existence of this effect suggests control of the mitochondrial oxidative phosphorylation by the cytoplasmic phosphorylation through the competition between pyruvate kinase and phosphorylation sites for ADP. Acknowledgments. This work was supported by a grant of the Comisi6n Asesora de Investigacien Cientlfica y TCcnica. The authors are grateful to Dr. J.M. Ramfrez for helpful criticism of the manuscript. Thanks are due to Miss ConcepciBn Baranda for technical assistance. REFERENCES l.2 .3.4 .-
Weinhouse, S. (1974) Control process in neoplasia, (M-A. Mechlman and R.W. Hanson eds.) pp l-30, Academic Press, New York. Cohen, L.H. and Noell, W.K. (1960) J. Neurochem. 5, 253-276. Hassinen, I. and Ylikahri, R.H. (1970) Biochem. Biophys. Res. comm., 38, 1091-1097. Chance, B. and Hess, B. (1959) Science, 129, 700-708.
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Vol. 83, No. 4, 1978
BIOCHEMICAL
AND BIOPHYSICAL
5 6
..-
Seglen, Berry,
0. (1974) Biochim. Biophys. M.N. and Friend, D.S. (1969)
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Cornell, N.W., Lund, P., Hems, Biochem. J. 134, 671-672. Wanneste, W.H. (1966) Biochim. Chance, B. and Hess, B. (1959)
RESEARCH COMMUNICATIONS
Acta, 338, 317-336. J. Cell Biol. 43,
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R. and Krebs,
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Woods, H.F., Eggleston, L.V. and Krebs, H.A. (1970) Biochem. J. 119, 501-510. Hassinen, 1-c Ylikahri, R.H. and Kahonen, M.T. (1971) Arch. Biochem. Biophys. 147, 255-261. B. and Hess, B. (1959) J. Biol. Chem. 234, 2416Chance, 2420.
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Gosblvez, M., P&ez-Garcia, J. and Weinhouse, S. (1974) Eur. J. Biochem. 46, 133-140. Sauer, L.A. (1977) J. Cell. Physiol. 93, 313-315. Sillero, M.A.G., Sillero, A. and Sols, A. (1969) Eur. J. Biochem. 10, 345-350. Moreno, Fx., Benito, M., Sbnchez-Medina, E., Medina, J. M. and Mayor, F. (1976) Mol. Cell. Biochem. 13, 89-93.
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