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

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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.

1427

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.

1428

Vol. 83, No. 4, 1978

BIOCHEMICAL

AND BIOPHYSICAL

5 6

..-

Seglen, Berry,

0. (1974) Biochim. Biophys. M.N. and Friend, D.S. (1969)

7

.-

8 9

..-

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,

506-520.

R. and Krebs,

H.A.

(1973)

Biophys. J. Biol.

113,

175-178.

Acta

Chem.

234,

2421-2427.

lO.ll.12.-

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.

13.14.-

15.16.-

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.

1429

Crabtree effect induced by fructose in isolated hepatocytes from fed rats.

BIOCHEMICAL Vol. 83, No. 4, 1978 AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages August 29,1978 1422-1429 CRABTREE EFFECT INDUCED BY FRUCTOSE IN...
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