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Activities of l -lactate and glycerol phosphate production rates in vitro from glucose 6-phosphate in regenerating rat liver
Int. J. Biochem. Vol. 17, No. 9, pp. 1015-1017, 1985 Printed in Great Britain. All rights reserved
0020-711X/85 $3.00 + 0.00 Copyright © 1985 Pergamon Press Ltd
ACTIVITIES OF L-LACTATE A N D GLYCEROL PHOSPHATE PRODUCTION RATES IN VITRO FROM GLUCOSE 6-PHOSPHATE IN R E G E N E R A T I N G RAT LIVER Jos~ M. SIVERIO, NIkSTOR V. TORRES and ENRIQUE MELI~NDEZ-HEVIA* Departamento de Bioquimica, Facultad de Biologia, Universidad de La Laguna, 38000 Tenerife, Canary Islands, Spain (Received 7 December 1984)
Abstract--1. Activities of L-lactate and glycerol phosphate production in vitro were assayed in regenerating rat liver at different times after partial hepatectomy and were compared with activities of sham operated controls. 2. Results show an oscillatory shift in these activities with smallest values for both L-lactate and glycerol phosphate production rates at 20 hr, these being 80~ and 50~, respectively of normal values, and with highest values at 42 hr where these rates were 150~ and 125~, respectively, both recovering their original state 60 hr after surgery. 3. From these data it is concluded that in regenerating liver there are changes in the ability of the glycolysis system to produce L-lactate and glycerol phosphate.
vitro during rat liver regeneration. Our results show
INTRODUCTION Rat liver regeneration after 7 0 ~ partial hepatectomy has been extensively studied as an experimental model of cell growth in controlled physiological conditions (see a review in Bresnick, 1971). Glucose homeostasis during early stages of rat liver regeneration has been studied by Petenusci et al. (1983); these authors conclude that the relatively high blood sugar levels during fasting in hepatectomized rats depend on a rapid increase in gluconeogenic activity, suggesting that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities. In fact, glucose metabolism during liver regeneration has been studied by several authors (see Eigenbrodt and Glossamn, 1980, for a review). Brinkman et al. (1978) have described an increase of phosphoenolpyruvate carboxikinase (E.C. 4.1.1.32) to over 2 0 0 ~ and a slight descent of pyruvate kinase (E.C. 2.7.1.40) during the first 72 hr after partial hepatectomy. However, changes in individual enzyme activities can be different to changes in total glycolysis activity since a complex metabolic pathway can be subjected to several regulation points. Thus, a slight variation of individual enzyme activity values can account for important changes in the activity of individual enzymes may not be manifest within the whole activity of the system, according to the relationship of each individual enzyme activity to the entire system (Kacser and Burns, 1979; Kacser, 1983; Heinrich and Rapoport, 1983; Groen et al., 1982). In a previous paper (Melrndez-Hevia et al., 1984) we have shown that there are significant differences between regulatory effects on individual glycolytic enzymes and the same effects on the total glycolytic activity in rat liver. Thus, the aim of this work is to assay the whole activity of the conversion of glucose 6-phosphate into L-lactate and glycerol phosphate in *To whom all correspondence should be addressed.
significant differences in these activities versus shamoperated controls, as a wave of glycolysis activity with a minimum at 20 hr and a maximum at 42 hr to up to 150~o of normal value for L-lactate, its normal activity being recovered 3 days after partial hepatectomy. MATERIALS AND M E T H O D S
Female Wistar albino rats (180-220 g) fed on a standard laboratory diet were used in all experiments. The animals were maintained under an inverted light-dark cycle of 12 hr with the dark period from 09.00 to 21.00 hr. Partial hepatectomies (70%) were carried out under ether anesthesia between 10.00-10.30hr as described by Higgins and Anderson (193 l). Sham-operated animals and non-operated animals were used as different controls. After surgery the animals were kept with food and water ad lib. until killing. At convenient times after surgery, according to the experiment, livers from three rats in the same conditions were obtained under ether anesthesia, cooled, chopped, pooled and homogenized at 1 g/3 ml in 0.1 M potassium phosphate buffer, pH 7.4, containing 5 mM NaC1 and 2.5 mM MgCI: by using a Potter-Elvejeim homogenizer with Teflon pestle in an ice-cold bath. The homogenates were clarified twice by centrifugation at 27,000g in a Sorvall RC-5B centrifuge at 3-4°C for 20 and 10 min, respectively. The resulting supernatants were used immediately for in vitro glycolysis assays and their protein concentrations were determined according to Lowry et al. (1951). Glycolysis activity assays from glucose 6-phosphate were carried out as previously described (Melrndez-Hevia et al., 1984) by incubation of liver extracts with 7mM glucose 6-phosphate, 15mM ADP, l m M ATP, 7mM NAD ÷, 0.3 mM cyclic AMP, 0.2 mM fructose 1,6-biphosphate and 0.5 mM 2,3-biphosphoglycerate in 0.1 M potassium phosphate buffer, pH 7.4 containing 5 mM NaCI and 2.5 MgC12. Tissue extracts were diluted in the same buffer to obtain about 5 protein mg/ml in the incubation mixture. Bacterial growth was avoided by addition of 1 mg/ml penicillin and 1 mg/ml streptomycin to incubation mixtures. Chicken egg white typsin inhibitor (Sigma, type II) was always added to incubation mixtures. The total volume of the incubation
1015
1016
Jos~ M. SIVERIOet al.
mixtures were 7 ml. Reactions were started by adding 0.1 ml of tissue extracts per ml of whole mixture and were carried out in a shaker bath at 30°C during 40 min, taking 1 ml aliquots every 5 min to assay L-lactate and glycerol phosphate. These aliquots were deproteinized by addition of 1 ml of 1M HC104, neutralized after 10min with 0.1ml of 5 M K2CO 3 (to pH 6-7) and clarified by centrifugation at 3500 x g for 10 min. L-lactate and glycerol phosphate were assayed in these extracts according to Bergmeyer (1974) by a continuous recording UV absorbance variation of NADH at 340 nm to end point with a Hitachi 100-80 A Spectrophotometer, at 25°C. All data were obtained from three experiments with each animal; means and coefficients of variation are given in Results. All biochemical reagents were obtained from Sigma Chem. Co. (St. Louis, MO, USA); all other reagents were obtained from E. Merck (Darmstadt, FRG), at analytical reagent grade. RESULTS
/
I
X
I
I
I
I
I
10
20
30
40
50
iI
1 100
Hours offer surgery
Fig. 2. Variation of activities for L-lactate production from glucose 6-phosphate in vitro in regenerating rat liver (11) and in liver of sham-operated controls (V]) at different times after surgery. Means _+ SD for three animals are given.
AND DISCUSSION
Figure 1 shows results of a typical kinetic experiment of L-lactate and glycerol phosphate production in vitro, from glucose 6-phosphate, by rat liver extract, during 40 min of incubation time. In these experiments good linearities both for L-lactate and for glycerol phosphate were obtained, allowing us to calculate specific activity values from the slopes of these curves. Data of Figs. 2 and 3 show specific activity values for L-lactate and glycerol phosphate production respectively, of regenerating and shamoperated rat livers. Normal specific activity of the rat liver of non-operated animals gives values of 6.6 + 0.5 # m o l of L-lactate per min, per protein mg and 6.4 + 0.5/~mol of glycerol phosphate per min, per protein mg, assayed in these conditions. It can be seen that changes in L-lactate and glycerol phosphate production rates after partial hepatectomy show a certain parallelism with the shifts in values of shamoperated controls, although in regenerating liver the changes are more dramatic. Partial hepatectomy and single sham-operation produce a decay in the activity of glycerol phosphate production in a first period of regeneration until 30 hr. This effect is also observed, although to a lesser degree in sham-operated controis, being more persistent in regenerating liver. In a subsequent time period, activity of glycerol phosphate production is slightly enhanced, its values in
5!
sE >
o.
L
.....
I0
20
30
Hours
ofter
40
50
100
surgery
Fig. 3. Variation of activities for glycerol phosphate production from glucose 6-phosphate in vitro in regenerating rat liver ( I ) and in liver of sham-operated controls (r-q) at different times after surgery. Means + SD for three animals are given. sham-operated controls recovering the normal value; moreover, activity of L-lactate production of regenerating livers shows a great increase, signicantly different from sham-operated controls. In normal non-operated animals, liver extracts give about the same activity for L-lactate and glycerol phosphate production rates but after surgery, in all cases, values of activity for L-lactate greater that for glycerol phosphate were obtained until 60 hr. After this time L-lactate and glycerol phosphate production returned to their normal values and their ratio was again about one. 16
--06
4
e
1.4
/'-o5 Tm~
3
•
"~ E2 o
/
~.1 Q
i IQ
Time
E
-o3
s
-0.2
~
o
-04
i 50
09
~ bo-
10,
S.
~a~
08
E
~806 _o5_
_
0 i :::L i 20
-..>
i 40
~
0.4 02
of incubation (min)
Fig. l. Results of a typical kinetic experiment showing L-lactate (0) and glycerol phosphate (O) production rates from glucose 6-phosphate by rat liver extract in vitro, according to procedure described under "Materials and Methods. Velocities were calculated from slopes of these curves whose linearities were maintained during 40min.
•10
20
50
40
50
100
Hours a f t e r surgery
Fig. 4. Variation of activity of regenerating rat liver to produce L-lactate ( 0 ) and glycerol phosphate (O) from glucose 6-phosphate in vitro at different times after surgery. Means + SD for three animals are given.
Glycolysis in regenerating rat liver Figure 4 represents the quotients among activities of regenerating and sham-operated rat livers for L-lactate and glycerol phosphate production rates. It can be seen that shifts of these activities from their normal values give waves for these two products with a maximal extension between 20 and 42 hr of regeneration time, and always with greater values for L-lactate than for glycerol phosphate. These waves persist during the first 60 hr, after which they recover their normal values. These data allow us to conclude that during rat liver regeneration, in the first 6 0 h r after partial hepatectomy, activity of glycolysis is submitted to changes which account for different ability to produce L-lactate and glycerol phosphate from glucose 6-phosphate. Acknowledgements--This work was supported by a research
grant from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica, Ref. No. 4119/79. REFERENCES
Bergmeyer H. U. (1974) Methods in Enzymatic Analysis, Vol. 3. Verlag Chemie/Academic Press, New York. Bresnick E. (1971) Regenerating liver: An experimental model for the study of growth. Methods Cancer Res. 6, 347-397. Brinkmann A., Katz N., Sasse D. and Jungermann K. (1978) Increase of the gluconeogenic and decrease of the glycolytic capacity of rat liver with a change of metabolic
1017
zonation after partial hepatectomy. Hoppe-Seyler's. Z. Physiol. Chem. 359, 1561-1571. Eigenbrodt E. and Glossamn H. (1980) Glycolysis-one of the keys to cancer? Trends pharmacol. Sci. 1, 240-245. Groen A. K., Van der Meer R., Westerhoff H. V., Wanders R. J. A., Akerboom T. P. M. and Tager J. M. (1982) Control of metabolic fluxes. In Metabolic Compartmentation (Edited by Sies H.) pp. 9-37. Academic Press, New York. Heinrich R. and Rapoport S. M. (1983) The utility of mathematical models for the understanding of metabolic systems. Biochem. Soc. Trans. 11, 31-35. Higgins G. M. and Anderson R. M. (1931) Experimental pathology of the liver I. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12, 186-202. Kacser H. (1983) The control of enzyme systems in vivo: Elasticity analysis of the steady state. Biochem. Soc. Trans. 11, 35-40. Kacser H. and Burns J. A. (1979) Molecular democracy: Who shares the Controls? Biochem. Soc. Trans. 7, 1149-1160.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mel6ndez-Hevia E., Siverio J. M. and P6rez J. A. (1984) Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity. Int. J. Biochem. 16, 469-476. Petenusci S. O., Freitas T. C., Roselino E. S. and Migliorini R. H. (1983) Glucose homeostasis during the early stages of liver regeneration in fasted rats. Can. J. Physiol. Pharmacol. 61, 222-228.
0020-711X/85 $3.00 + 0.00 Copyright © 1985 Pergamon Press Ltd
ACTIVITIES OF L-LACTATE A N D GLYCEROL PHOSPHATE PRODUCTION RATES IN VITRO FROM GLUCOSE 6-PHOSPHATE IN R E G E N E R A T I N G RAT LIVER Jos~ M. SIVERIO, NIkSTOR V. TORRES and ENRIQUE MELI~NDEZ-HEVIA* Departamento de Bioquimica, Facultad de Biologia, Universidad de La Laguna, 38000 Tenerife, Canary Islands, Spain (Received 7 December 1984)
Abstract--1. Activities of L-lactate and glycerol phosphate production in vitro were assayed in regenerating rat liver at different times after partial hepatectomy and were compared with activities of sham operated controls. 2. Results show an oscillatory shift in these activities with smallest values for both L-lactate and glycerol phosphate production rates at 20 hr, these being 80~ and 50~, respectively of normal values, and with highest values at 42 hr where these rates were 150~ and 125~, respectively, both recovering their original state 60 hr after surgery. 3. From these data it is concluded that in regenerating liver there are changes in the ability of the glycolysis system to produce L-lactate and glycerol phosphate.
vitro during rat liver regeneration. Our results show
INTRODUCTION Rat liver regeneration after 7 0 ~ partial hepatectomy has been extensively studied as an experimental model of cell growth in controlled physiological conditions (see a review in Bresnick, 1971). Glucose homeostasis during early stages of rat liver regeneration has been studied by Petenusci et al. (1983); these authors conclude that the relatively high blood sugar levels during fasting in hepatectomized rats depend on a rapid increase in gluconeogenic activity, suggesting that hepatocytes in remnant liver can proliferate under conditions of maximal gluconeogenic and low glycolytic activities. In fact, glucose metabolism during liver regeneration has been studied by several authors (see Eigenbrodt and Glossamn, 1980, for a review). Brinkman et al. (1978) have described an increase of phosphoenolpyruvate carboxikinase (E.C. 4.1.1.32) to over 2 0 0 ~ and a slight descent of pyruvate kinase (E.C. 2.7.1.40) during the first 72 hr after partial hepatectomy. However, changes in individual enzyme activities can be different to changes in total glycolysis activity since a complex metabolic pathway can be subjected to several regulation points. Thus, a slight variation of individual enzyme activity values can account for important changes in the activity of individual enzymes may not be manifest within the whole activity of the system, according to the relationship of each individual enzyme activity to the entire system (Kacser and Burns, 1979; Kacser, 1983; Heinrich and Rapoport, 1983; Groen et al., 1982). In a previous paper (Melrndez-Hevia et al., 1984) we have shown that there are significant differences between regulatory effects on individual glycolytic enzymes and the same effects on the total glycolytic activity in rat liver. Thus, the aim of this work is to assay the whole activity of the conversion of glucose 6-phosphate into L-lactate and glycerol phosphate in *To whom all correspondence should be addressed.
significant differences in these activities versus shamoperated controls, as a wave of glycolysis activity with a minimum at 20 hr and a maximum at 42 hr to up to 150~o of normal value for L-lactate, its normal activity being recovered 3 days after partial hepatectomy. MATERIALS AND M E T H O D S
Female Wistar albino rats (180-220 g) fed on a standard laboratory diet were used in all experiments. The animals were maintained under an inverted light-dark cycle of 12 hr with the dark period from 09.00 to 21.00 hr. Partial hepatectomies (70%) were carried out under ether anesthesia between 10.00-10.30hr as described by Higgins and Anderson (193 l). Sham-operated animals and non-operated animals were used as different controls. After surgery the animals were kept with food and water ad lib. until killing. At convenient times after surgery, according to the experiment, livers from three rats in the same conditions were obtained under ether anesthesia, cooled, chopped, pooled and homogenized at 1 g/3 ml in 0.1 M potassium phosphate buffer, pH 7.4, containing 5 mM NaC1 and 2.5 mM MgCI: by using a Potter-Elvejeim homogenizer with Teflon pestle in an ice-cold bath. The homogenates were clarified twice by centrifugation at 27,000g in a Sorvall RC-5B centrifuge at 3-4°C for 20 and 10 min, respectively. The resulting supernatants were used immediately for in vitro glycolysis assays and their protein concentrations were determined according to Lowry et al. (1951). Glycolysis activity assays from glucose 6-phosphate were carried out as previously described (Melrndez-Hevia et al., 1984) by incubation of liver extracts with 7mM glucose 6-phosphate, 15mM ADP, l m M ATP, 7mM NAD ÷, 0.3 mM cyclic AMP, 0.2 mM fructose 1,6-biphosphate and 0.5 mM 2,3-biphosphoglycerate in 0.1 M potassium phosphate buffer, pH 7.4 containing 5 mM NaCI and 2.5 MgC12. Tissue extracts were diluted in the same buffer to obtain about 5 protein mg/ml in the incubation mixture. Bacterial growth was avoided by addition of 1 mg/ml penicillin and 1 mg/ml streptomycin to incubation mixtures. Chicken egg white typsin inhibitor (Sigma, type II) was always added to incubation mixtures. The total volume of the incubation
1015
1016
Jos~ M. SIVERIOet al.
mixtures were 7 ml. Reactions were started by adding 0.1 ml of tissue extracts per ml of whole mixture and were carried out in a shaker bath at 30°C during 40 min, taking 1 ml aliquots every 5 min to assay L-lactate and glycerol phosphate. These aliquots were deproteinized by addition of 1 ml of 1M HC104, neutralized after 10min with 0.1ml of 5 M K2CO 3 (to pH 6-7) and clarified by centrifugation at 3500 x g for 10 min. L-lactate and glycerol phosphate were assayed in these extracts according to Bergmeyer (1974) by a continuous recording UV absorbance variation of NADH at 340 nm to end point with a Hitachi 100-80 A Spectrophotometer, at 25°C. All data were obtained from three experiments with each animal; means and coefficients of variation are given in Results. All biochemical reagents were obtained from Sigma Chem. Co. (St. Louis, MO, USA); all other reagents were obtained from E. Merck (Darmstadt, FRG), at analytical reagent grade. RESULTS
/
I
X
I
I
I
I
I
10
20
30
40
50
iI
1 100
Hours offer surgery
Fig. 2. Variation of activities for L-lactate production from glucose 6-phosphate in vitro in regenerating rat liver (11) and in liver of sham-operated controls (V]) at different times after surgery. Means _+ SD for three animals are given.
AND DISCUSSION
Figure 1 shows results of a typical kinetic experiment of L-lactate and glycerol phosphate production in vitro, from glucose 6-phosphate, by rat liver extract, during 40 min of incubation time. In these experiments good linearities both for L-lactate and for glycerol phosphate were obtained, allowing us to calculate specific activity values from the slopes of these curves. Data of Figs. 2 and 3 show specific activity values for L-lactate and glycerol phosphate production respectively, of regenerating and shamoperated rat livers. Normal specific activity of the rat liver of non-operated animals gives values of 6.6 + 0.5 # m o l of L-lactate per min, per protein mg and 6.4 + 0.5/~mol of glycerol phosphate per min, per protein mg, assayed in these conditions. It can be seen that changes in L-lactate and glycerol phosphate production rates after partial hepatectomy show a certain parallelism with the shifts in values of shamoperated controls, although in regenerating liver the changes are more dramatic. Partial hepatectomy and single sham-operation produce a decay in the activity of glycerol phosphate production in a first period of regeneration until 30 hr. This effect is also observed, although to a lesser degree in sham-operated controis, being more persistent in regenerating liver. In a subsequent time period, activity of glycerol phosphate production is slightly enhanced, its values in
5!
sE >
o.
L
.....
I0
20
30
Hours
ofter
40
50
100
surgery
Fig. 3. Variation of activities for glycerol phosphate production from glucose 6-phosphate in vitro in regenerating rat liver ( I ) and in liver of sham-operated controls (r-q) at different times after surgery. Means + SD for three animals are given. sham-operated controls recovering the normal value; moreover, activity of L-lactate production of regenerating livers shows a great increase, signicantly different from sham-operated controls. In normal non-operated animals, liver extracts give about the same activity for L-lactate and glycerol phosphate production rates but after surgery, in all cases, values of activity for L-lactate greater that for glycerol phosphate were obtained until 60 hr. After this time L-lactate and glycerol phosphate production returned to their normal values and their ratio was again about one. 16
--06
4
e
1.4
/'-o5 Tm~
3
•
"~ E2 o
/
~.1 Q
i IQ
Time
E
-o3
s
-0.2
~
o
-04
i 50
09
~ bo-
10,
S.
~a~
08
E
~806 _o5_
_
0 i :::L i 20
-..>
i 40
~
0.4 02
of incubation (min)
Fig. l. Results of a typical kinetic experiment showing L-lactate (0) and glycerol phosphate (O) production rates from glucose 6-phosphate by rat liver extract in vitro, according to procedure described under "Materials and Methods. Velocities were calculated from slopes of these curves whose linearities were maintained during 40min.
•10
20
50
40
50
100
Hours a f t e r surgery
Fig. 4. Variation of activity of regenerating rat liver to produce L-lactate ( 0 ) and glycerol phosphate (O) from glucose 6-phosphate in vitro at different times after surgery. Means + SD for three animals are given.
Glycolysis in regenerating rat liver Figure 4 represents the quotients among activities of regenerating and sham-operated rat livers for L-lactate and glycerol phosphate production rates. It can be seen that shifts of these activities from their normal values give waves for these two products with a maximal extension between 20 and 42 hr of regeneration time, and always with greater values for L-lactate than for glycerol phosphate. These waves persist during the first 60 hr, after which they recover their normal values. These data allow us to conclude that during rat liver regeneration, in the first 6 0 h r after partial hepatectomy, activity of glycolysis is submitted to changes which account for different ability to produce L-lactate and glycerol phosphate from glucose 6-phosphate. Acknowledgements--This work was supported by a research
grant from the Comisi6n Asesora de Investigaci6n Cientifica y T6cnica, Ref. No. 4119/79. REFERENCES
Bergmeyer H. U. (1974) Methods in Enzymatic Analysis, Vol. 3. Verlag Chemie/Academic Press, New York. Bresnick E. (1971) Regenerating liver: An experimental model for the study of growth. Methods Cancer Res. 6, 347-397. Brinkmann A., Katz N., Sasse D. and Jungermann K. (1978) Increase of the gluconeogenic and decrease of the glycolytic capacity of rat liver with a change of metabolic
1017
zonation after partial hepatectomy. Hoppe-Seyler's. Z. Physiol. Chem. 359, 1561-1571. Eigenbrodt E. and Glossamn H. (1980) Glycolysis-one of the keys to cancer? Trends pharmacol. Sci. 1, 240-245. Groen A. K., Van der Meer R., Westerhoff H. V., Wanders R. J. A., Akerboom T. P. M. and Tager J. M. (1982) Control of metabolic fluxes. In Metabolic Compartmentation (Edited by Sies H.) pp. 9-37. Academic Press, New York. Heinrich R. and Rapoport S. M. (1983) The utility of mathematical models for the understanding of metabolic systems. Biochem. Soc. Trans. 11, 31-35. Higgins G. M. and Anderson R. M. (1931) Experimental pathology of the liver I. Restoration of the liver of the white rat following partial surgical removal. Arch. Pathol. 12, 186-202. Kacser H. (1983) The control of enzyme systems in vivo: Elasticity analysis of the steady state. Biochem. Soc. Trans. 11, 35-40. Kacser H. and Burns J. A. (1979) Molecular democracy: Who shares the Controls? Biochem. Soc. Trans. 7, 1149-1160.
Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265-275. Mel6ndez-Hevia E., Siverio J. M. and P6rez J. A. (1984) Studies on glycolysis in vitro: role of glucose phosphorylation and phosphofructokinase activity on total velocity. Int. J. Biochem. 16, 469-476. Petenusci S. O., Freitas T. C., Roselino E. S. and Migliorini R. H. (1983) Glucose homeostasis during the early stages of liver regeneration in fasted rats. Can. J. Physiol. Pharmacol. 61, 222-228.