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The absence of dietary regulation of hepatic pyruvate kinase in the domestic fowl
fnt. 3. f%ochem., 1971, z,
271-275.
THE ABSENCE PYRUVATE
[Scientechnica
OF DIETARY KINASE
(PubIishers)
Ltd.]
271
REGULATION
OF HEPATIC
IN THE DOMESTIC J.
FOWL
PEARCE
Department of Agricultural Chtmistry, Queen’s University, Belfast, and Ministry of Agriculture, Northern Ireland (Received8 Jtdy, I 970) ABSTRACT I. Diets containing 60 per cent, 70 per cent, and 80 per cent glucose or fructose or 18 per cent lipid were fed to pullets for varying periods of time. 2. No variation in the specific activity of pyruvate kinase was observed following these dietary treatments. 3. The experiment was also repeated with rats where variation in the specific activity of this enzyme was readily shown. 4. The resultsare discussedin relation to the mechanismsof control of pyruvate kinase known to operate in mammals.
THE role of pyruvate
kinase with respect to the regulation of glycolysis and gluconeogenesis is well documented (Weber, Lea, Convery, and Stamm, 1967). The synthesis of this enzyme is induced by high-carbohydrate diets and is repressed by feeding a low-carbohydrate, high-protein diet (Krebs
and Eggleston, I 965 ; Freedland, Cunliffe, and Zinkl, 1966; Szepesi and Freedland, rg68a). The type of dietary carbohydrate also affects the specific activity of pyruvate kinase. Fructose fed at the same dietary concentration as glucose has been shown to cause a greater activity of this enzyme in the rat (Bailey, Taylor, and Bartley, x968; Szepesi and Freedfand, rg68a, b). Sucrose feeding also induces increased pyruvate kinase activity (Krebs and Eggleston, 1965; Bailey, Taylor, and Bartley, 1968; Szepesi and Freedland, rg68a), which suggests that the increase is due to the fructose moiety of sucrose. During experiments on the effects of dietary variation on enzyme activity in the domestic fowl, it was noted that pyruvate kinase did not exhibit any significant variation in specific activity between treatments. This paper describes experiments carried out to examine the phenomenon of non-adaptation of pyruvate kinase in the domestic fowl.
MATERIALS
AND METHODS
The pullets used were white Leghorns (Gaily busts) and the rats (R&us nonneginrr) employed were Lister hooded rats fkom the departmental colony. EXPERIMENTI Six-week-old pullets were maintained under conditions of continuous artificial light at a temperature of 20-2 x0C. On entering the animal house, the birds were allowed at least 4 days on a commercial rearing diet to allow accli;na&zation and familiarization with the environment. The birds were then randomized by weight and placed into 5 treatment groups; eaih treatment -group received a different dietarv m&me. Diet I. the control diet, was a cert&ba&d rearing &sh consisting of 169 per cent protein, 3.84 per cent oif, 2-03 per cent fibre, 5.65 per cent ash, o-go per cent calcium, and 0.60 per cent phosphorus. Diet 2 contained go per cent fructose, ro per cent casein, 3 per cent gelatin, 6.0 per cent mineral salts mixture, o-3 per cent vitamin mixture, 0.3 per cent cystine, 0.2 per cent choline chloride, 0.1 per cent tryptophan, and 0.1 per cent inositol. The composition of the mineral salts and vitamin mixtures are described by Balnave and Brown ( 1967). Diet 3 contained 60 ner cent glucose, _ 22 per cent c&in, and II per-cent gelahn; the rest of the diet had the same comnosition as Diet 2. In Diets 4 and 5, glucose replaced fructose at the 80 ner cent and 60 ner cent con&ntrations as in diets 2 and 3 respec~tively. Each group of birds was supplied with food and water ad lib. and fed for a period of 4 days with the experimental diets.
272
PEARCE
tiPERXMENT2
Three-month-old femaIe rats were housed individually and randomized by weight into 5 experimental groups. The animals were maintained under conditions of continuous artificial light at 2o-2 I’ C. Each group received one of the diets described for Experiment I for a period of 4 days. The rats were provided with food and water ad lib. throughout the experiment. EXPERIMENT3
One-day-old pullets were divided into 3 experimental groups and placed in a brooder at 32-35” C. Each group received a different dietary regime and food and water were provided ad lib. Diet I was a cereal-baaed rearing mash, I)iet 2 contained 70 per cent fructose, and Diet 3 contained 7o per cent glucose. The full comnositions of these diets have-been described previ&sly (Pearce, 1970). At 1 weeks of age the birds were nlaced in individual cag& at a tekperature of 2&21O C. and under conditions of continuous artificial light. The 3 experimental treatments were randomized throughout the cages. The experimental diets were provided for a further week until the birds were 4 weeks old. EXI”E~GMENT 4
One-month-old female rats were &aced in 3 experimental groups, housed in &&dual cagtts: and nrovided with food and water ad lib. Each expehmental group was provided with one of the diets described in Experiment 3 for a period of 4 weeks. EXPER~IUENT 5 Twenty-two-week-old laying hens were randomized into 2 experimental treatments, housed in individual cages, and provided with food and
water ad I&. One group received a lipid-supplemented diet and the other group received a cerealbased control diet. The high-oil diet contained 20.58 per cent protein, I 7.75 per cent oil, 2.7 I per cent fibre, 9.05 per cent ash, 3.91 per cent Ca, and 0.76 Der cent P: the oil was nrovided as arachis’oil: The con& diet containid x3.97 per cent protein, 1.~4 per cent oil, 3.33 per cent fibre, 8.23 per cent ash, 4’04 per cent Ca, and 0.64 per cent P. The birds were maintained on these diets for a further 52 weeks and thus were 74 weeks old when killed. At the end of the period of feeding the birds were killed bv decaoitation and the rats bv cervical
dislocation. ‘Their‘ livers were rapidly iemoved, chilled, and portions homogenized with 4 volumes of ice-cold 0.1 M potassium phosphate buffer, #-I 7.0, containing 7.0 m&f 2-mercaptoethanol. The homogenates were centrifuged at xoo,ooog for I hour at o-4* C. (MSE supenpeed so), and the resulting supematant assayed for pyruvate kinase activity. The activity of pyruvate kinase (B.C. 2.7.1.40) was assayed by determining the
ht.
3. Biockmz.
rate of formation of pyruvate s~ctrophotome~itally in the presence of lactic dehydrogenase (B&her and Pfieiderer, 1955). Identical cuvettes were made up containing, in umoles: potassium phosphate buffer, PI-I 7.0, 200; M&I, IO; KC1 50; ADP 2.53 20 ~1. lactic dehydrogenase (equivalent 30 I.U.) ; and liver extract in a total volume of 2.7 ml. NADH (0.3 &mole) was added to the experimental cuvette and after the initial measurement of NADH dehydrogenase activity in the extract, Q fimoles phosphoenol pymvate were added and the decrease in absorption at 340 rng determined. The assays were carried out in a Unicam SP 800 recording spectrophotometer at 40” C. and in all cases the recorded activity was linear with respect to both time and protein
concentration in the extract. The protein content of cell-free extracts was determined by the biuret reaction (Layne, I957). RESULTS The results in Table I show the effects of feeding glucose and fructose for 4 days on the activity of hepatic pyruvate kinase (Experiment I) and it can be seen that there was no significant variation in the specific activity of the chick enzyme between dietary treatments. This experiment was also carried out using rats (Experiment 2) since in rats pyruvate kinase has been shown to vary in activity according to the type of carbohydrate in the diet. It was found that there was a difference between the specific activity in fructose-fed animals and in those receiving the other dietary treatments (Table I). Also given in Table I are some results from an earlier inv~tigation (Pearce, 1970) which similarly show that there was no significant variation of pyruvate kinase activity between treatments when diets containing 70 per cent fructose or 7o per cent glucose were fed to 6-week-old pullets for 4 days. Longer-term experiments were carried out in which the animals were on the experimental diets for 4 weeks (Table II). Again, no significant differences in the specific activities of chick pyruvate kinase were found between dietary treatments whereas in the rat the activity was greatest in the fructosefed animals (experiment 3 and 4). The effects of long-term fat feeding on hepatic pyruvate kinase activity in the adult laying hen (Experiment 5) are also shown in Table II. The results show that the dietary fat
50.6 fs.97 (4) rsg4.of 152.1 (6)
go per cent Fructose
46.7 rfr5.55 (5)* -
--
70 per cent Fructose 5r.29f5.87 673.1 fg4.9
60 per cent Fructose (4) (3)
47’43f7.07 548rt63.6
(4) (3)
80 per cent Glucose -_I____
DUET
(5)*
gr.34f7.r~ 1528.5 (2)
(6) g46of5*32 639-3 (2)
(6)
70 per cent Glucose g3.74f6.87 697.ofa4.r
(6) (3)
Cereal-based Control Diet
’
58.6f6.4 -
(3)
High Fat
Enzyme activities given as mpmoles per minute per mg. protein in the extract&S.E.M. of observations is given in parentheses.
-Chicken Rat
ANIMALS
RXET
(3)
The number
6a.4f3.6 -
Low Fat Control
Table I&--EFFECTSOF LONG-TERM FEEDING OFDIETSCONTASNING VARIOUSPROPORTIONS OFCARBOHYDRATE ORFAT ONTWESPECIFICACZT~V~TY OF HEPATK ~YRUVATEKINA~EINTHECHIC~CEN AND THE: RAT
EXPERIMENTAL
56.4r f7.or (4) 4g6*4f ~4’6 (6)
Control --
PYRUV~TE
is given in parentheses.
55’51 f5’2 1 (4) 468.4f49.2 (3)
60 per cent Glucose
The number olobservations
55’3f5.05 -
70 per cent Glucose
OF FEEDma DIETSCONTAINING DIFFERENTPNoeorwroNs OF GLWXE AND FRUCTOSEFOR 4 DAYS ON THE HEPATIC KINAS AortvfrY~~ THECH~CKEN ANDTHE RAT
Enzyme activities given as mumoles per minute per mg. protein in the extractfS.E.M. * These results taken from Pearce (1970).
Chicken Rat
EXPERIMENTAL ANIMAL
Table I.-EFFECTS
PEARCE
271
had no significant effect on the specific activity of this enzyme. It has been shown that long-term fat feeding significantly affects the activities of the lipogenic enzymes in the liver of the laying hen (Pearce, 1968) and this indicates that the non-adaptation of the pyruvate kinase is a true observation and is not an artefact due to the method of extract preparation. DISCUSSION
The results reported in this paper support 1970) that earlier observations (Pearce, pyruvate kinase activity in the domestic fowl is not significantly affected by diet. This observation was unexpected since pyruvate kinase is considered to be a ratelimiting glycolytic enzyme and has been shown to be susceptible to dietary manipulation. High-carbohydrate diets and particular diets containing fructose have been shown to increase the specific activity of hepatic pyruvate kinase in the rat (Krebs and Eggleston, 1965 ; Tanako, Harano, Sue, and Morimura, I 967 ; Bailey, Taylor, and Bartley, 1968; Szepesi and Freedland, 1g68a, b). In the present work the rat was used to obtain a comparison under the same experimental conditions and it was found that variation of pyruvate kinase activity between dietary treatments was readily detectable and showed the same pattern of activity as had been obtained by other workers. It is noteworthy that the specific activity of the enzyme was of the same order as that reported by Krebs and Eggleston (I 965) and Bailey, Taylor, and Bartley ( I 968). These results indicate that the techniques employed of tissue extract preparation and enzyme assay were capable of showing variation of enzyme specific activity in the rat which is known to exhibit the phenomenon and so the observed nonadaptation of pyruvate kinase activity in the domestic fowl was not an artefact. Further evidence that these results were not due to poor experimental technique was the observation that significant alterations of ketohexokinase activity were detected in the same extracts which show non-adaptation of pyruvate kinase (Pearce, 1970).
Dietary fat is also known to have an effect on pyruvate kinase activity in the rat. During the suckling period when the diet contains a high proportion of fat the specific activity of this enzyme is low compared with the postsuckling period and, similarly, placing rats from a low-fat diet on to one supplemented with fat results in a repression of pyruvate kinase activity (Taylor, Bailey, and Bartfey, I 967). The effect of dietary lipid on pyruvate kinase activity in the domestic fowl was exaciined in this experiment and again no variation in enzyme specific activity was found between dietary treatments. In contrast to the situation in the rat, mouse liver pyruvate kinase shows very little alteration in activity with changes in diet (Bailey, Taylor, and Bartley, 1968) and Gevers (1967) found that the enzyme in pigeon liver does not exhibit any variation in activity on starvation and refeeding. The situation in the domestic fowl would appear to be analogous to that in the pigeon and it seems probable that the activity of pyruvate kinase is not controlled at the level of enzymeprotein synthesis but at the level of interaction of the preformed enzyme with inhibitors (ATP, Cusf, alanine, proline, NADH, free fatty acids, ace+-CoA, and oxaloacetate) and activators (fructose- I ,6diphosphate, fructose- I -phosphate, glucose6-phosphate, and glyceraldehyde-S-phosphate) (Tanako and others, 1967 ; Taylor and Bailey, 1967 ; Bailey, Stirpe, and Taylor, 1968; Seubert, Henning, Schoner, and L’Age, 1968; Weber, Lea, and Stamm, 1968; Eggleston and Woods, 1970). Leveille ( 19%) has recently obtained evidence to show that pyruvate kinase from the liver of the chicken is allosterically activated by fructose-1,6diphosphate and this supports the view that regulation is effected at the level of preexisting enzyme. SUMiMARY I. The effect of dietary fructose, glucose, and lipid on hepatic pyruvate kinase activity in the domestic fowl was investigated. 2. Immature pullets were fed diets containing either 60, 70, or 80 per cent glucose or fructose or a cereal-based control diet for
‘97’,2
PYRUVATE KlNASEINDEPENDENT OF DIET
periods of 4 days and 4 weeks. Diets containing I 8 per cent dietary lipid or a cereal-based layers mash were fed to laying hens for 52 weeks 3. It was found that there was no significant variation in the specific activity of hepatic pyruvate kinase between dietary treatments. 4. The experiments were repeated using rats, and variation in specific activity of the enzyme between dietary treatments was readily detectable. 5. The results are discussed in relation to the mechanisms of control of pyruvate kinase which are known to operate in mammals. ACKNOWLEDGEMENTS The author wishes to thank the British Egg Marketing Board for the award of a Post-doctoral Research Fellowship, during the tenure of which part of this work was carried out. He also thanks Mr. E. Wallace for technical assistance. REFERENCES BAILEY,E., STIRPE,F., and TAYLOR,C. B. (1g68), ‘ Reaulation of rat-liver Dvruvate kinase. The effe; of preincubation pH,‘copper ions, fmctose diphosphate and dietary changes on enzyme activity ‘, Biochem. J., x08,417-436. BAILEY, I$ TAYLOR, C. B., and BARTLEY, W. (1968)9 Effect of dietary carbohydrate on hepatic lipogenesis in the rat ‘, Nature, Lond., 2x7,471-47= BBCWER,T., and PFL.EIDERER, G. (x955), ‘ Pyruvate kinase from muscle ‘, in Metfwds in Enrymology (ed. Co~ownx, S. P., and KAPLAN,N. a.), vol. I,,__ w. 4’35-640. *__ ._ New York : Academic Press. EGGLE~T~N,L. V., and Wools, H. F. (rg7o), ’ Activation of liver pyruvate kinase by fructcuex-phosphate ‘, FEB.9 L.etters, 6,43-45. FREEDLAXD,R. A., CUNLIFFE,T. L., and ZINKL, J. G. (1g66), ‘ The effect of insulin on enzyme adaptations to diets and hormones‘, J. bial. Chem., 41,54.48-545 I. GEVERB,W. (1g67), ‘ The regulation of phosphoenolpyruvate synthesis in pigeon liver ‘, Biachem. 39 103, 141-152. KREBS, H. A., and EGGLESTON, L. V. (1g65), ‘ The role of pyruvate kinase in the regulation of gluconeogenesis ‘, &o&m. j., w 3G4C. LAY% E. (1957), ’ Spectrophotometric and turbidimetric methods for measuring proteins.
275
III. Biuret method ‘, in Methods in Enzpnolopl (ed. COLOWICK, S. P.,and KAPLAN,N.O.), vol. 3, pp. 450-451. New York: Academic Press. LEVEILLE.G. A. CI c&al. ‘ The influence of fructose diphosphatk &“pyruvate kinase activity in liver, muscle and adipose tissueof the rat, mouse, pig and chicken ‘, Camp. Biochem. Physiol., 28, 733-74o* PEARCE,J. (1g68), unpublished data. PEARCE,1. (Iqm), _. ., ‘ The effects of dietary fructose and ghxose on hepatic lipogenesis in the domestic fowl ‘. ht. 7. Biackm.. I. %06-st12. SEUBERT, W., HENNING:H. V., S&&R, W., and L’AGE, M. (1g68), ‘ Effects of cortisol on the levels of metabolites and enzymes controlling glucose production from pyruvate ‘, in Advances in Enzyme Regulation (ed. WEBER, G.), vol. 6, pp. I 53-180. London and New York : Pergamon. SZEPESI,B., and FREEDLAND,R. A. (1g68a), ’ Dietary regulation of pyruvate kinase synthesis in rat liver ‘, 3.JVutr., 9% 591-602. SZEPESI,B., and FREEDLAND,R. A. ( xg68b), ‘ Time course of enzyme adaptation. I. Effects of substituting dietary glucose and fructose at constant concentration of dietary protein ‘, Can. 3.B&x&m., 46, x45g-1470. TANAKO. T.. HARANO. Y., SUE. F., and MORIMURA,‘H.(I g67), ‘ C&&llization, ‘characterization and metabolic reguiation of two types of pyruvate kinase isolated from rat tissues ‘, 3. Bidsem., Taki, 6a, 71-91. TAYLOR,C. B., and BAILEY,E. (rg67), ‘ Activation of liver pyruvate kinase by fiustose-~$-diphosphate ‘, Biochem. J., 102, 32G33c. TAYLOR, C. B., BAILEY,E., and BARTLEY, W. ( Ig67), ‘ Changes in hepatic lipogenesis during development of the rat ‘, B&hem. J., 10%717722. WEBER,G., LEA, M. A., CONVERY, H. J. H., and STAMM,N. B. (1967), ‘ Regulation of gluconeogenesis and glycolysis; studies of mechanisms controlling enzyme activity ‘, in Adnames in Enryme apron (ed. WEBER, G.), vol. 5, London and New York: - 257-298. $Ergamon. WEBER, G., LEA, M. A., and ST-M, N. B. ( 1g68), ’ Sequential feedback inhibition and regulation of liver carbohydrate metabolism through control of enzyme activity ‘, in Advances in Enzpne Regulation (ed. W~BER, G.), vol. 6, pp101-123. London and New York: Pergamon. Kg Word Index: Dietary glucose, dietary fructose, dietary lipid, pyruvate kinase, rat, domestic fowl.
271-275.
THE ABSENCE PYRUVATE
[Scientechnica
OF DIETARY KINASE
(PubIishers)
Ltd.]
271
REGULATION
OF HEPATIC
IN THE DOMESTIC J.
FOWL
PEARCE
Department of Agricultural Chtmistry, Queen’s University, Belfast, and Ministry of Agriculture, Northern Ireland (Received8 Jtdy, I 970) ABSTRACT I. Diets containing 60 per cent, 70 per cent, and 80 per cent glucose or fructose or 18 per cent lipid were fed to pullets for varying periods of time. 2. No variation in the specific activity of pyruvate kinase was observed following these dietary treatments. 3. The experiment was also repeated with rats where variation in the specific activity of this enzyme was readily shown. 4. The resultsare discussedin relation to the mechanismsof control of pyruvate kinase known to operate in mammals.
THE role of pyruvate
kinase with respect to the regulation of glycolysis and gluconeogenesis is well documented (Weber, Lea, Convery, and Stamm, 1967). The synthesis of this enzyme is induced by high-carbohydrate diets and is repressed by feeding a low-carbohydrate, high-protein diet (Krebs
and Eggleston, I 965 ; Freedland, Cunliffe, and Zinkl, 1966; Szepesi and Freedland, rg68a). The type of dietary carbohydrate also affects the specific activity of pyruvate kinase. Fructose fed at the same dietary concentration as glucose has been shown to cause a greater activity of this enzyme in the rat (Bailey, Taylor, and Bartley, x968; Szepesi and Freedfand, rg68a, b). Sucrose feeding also induces increased pyruvate kinase activity (Krebs and Eggleston, 1965; Bailey, Taylor, and Bartley, 1968; Szepesi and Freedland, rg68a), which suggests that the increase is due to the fructose moiety of sucrose. During experiments on the effects of dietary variation on enzyme activity in the domestic fowl, it was noted that pyruvate kinase did not exhibit any significant variation in specific activity between treatments. This paper describes experiments carried out to examine the phenomenon of non-adaptation of pyruvate kinase in the domestic fowl.
MATERIALS
AND METHODS
The pullets used were white Leghorns (Gaily busts) and the rats (R&us nonneginrr) employed were Lister hooded rats fkom the departmental colony. EXPERIMENTI Six-week-old pullets were maintained under conditions of continuous artificial light at a temperature of 20-2 x0C. On entering the animal house, the birds were allowed at least 4 days on a commercial rearing diet to allow accli;na&zation and familiarization with the environment. The birds were then randomized by weight and placed into 5 treatment groups; eaih treatment -group received a different dietarv m&me. Diet I. the control diet, was a cert&ba&d rearing &sh consisting of 169 per cent protein, 3.84 per cent oif, 2-03 per cent fibre, 5.65 per cent ash, o-go per cent calcium, and 0.60 per cent phosphorus. Diet 2 contained go per cent fructose, ro per cent casein, 3 per cent gelatin, 6.0 per cent mineral salts mixture, o-3 per cent vitamin mixture, 0.3 per cent cystine, 0.2 per cent choline chloride, 0.1 per cent tryptophan, and 0.1 per cent inositol. The composition of the mineral salts and vitamin mixtures are described by Balnave and Brown ( 1967). Diet 3 contained 60 ner cent glucose, _ 22 per cent c&in, and II per-cent gelahn; the rest of the diet had the same comnosition as Diet 2. In Diets 4 and 5, glucose replaced fructose at the 80 ner cent and 60 ner cent con&ntrations as in diets 2 and 3 respec~tively. Each group of birds was supplied with food and water ad lib. and fed for a period of 4 days with the experimental diets.
272
PEARCE
tiPERXMENT2
Three-month-old femaIe rats were housed individually and randomized by weight into 5 experimental groups. The animals were maintained under conditions of continuous artificial light at 2o-2 I’ C. Each group received one of the diets described for Experiment I for a period of 4 days. The rats were provided with food and water ad lib. throughout the experiment. EXPERIMENT3
One-day-old pullets were divided into 3 experimental groups and placed in a brooder at 32-35” C. Each group received a different dietary regime and food and water were provided ad lib. Diet I was a cereal-baaed rearing mash, I)iet 2 contained 70 per cent fructose, and Diet 3 contained 7o per cent glucose. The full comnositions of these diets have-been described previ&sly (Pearce, 1970). At 1 weeks of age the birds were nlaced in individual cag& at a tekperature of 2&21O C. and under conditions of continuous artificial light. The 3 experimental treatments were randomized throughout the cages. The experimental diets were provided for a further week until the birds were 4 weeks old. EXI”E~GMENT 4
One-month-old female rats were &aced in 3 experimental groups, housed in &&dual cagtts: and nrovided with food and water ad lib. Each expehmental group was provided with one of the diets described in Experiment 3 for a period of 4 weeks. EXPER~IUENT 5 Twenty-two-week-old laying hens were randomized into 2 experimental treatments, housed in individual cages, and provided with food and
water ad I&. One group received a lipid-supplemented diet and the other group received a cerealbased control diet. The high-oil diet contained 20.58 per cent protein, I 7.75 per cent oil, 2.7 I per cent fibre, 9.05 per cent ash, 3.91 per cent Ca, and 0.76 Der cent P: the oil was nrovided as arachis’oil: The con& diet containid x3.97 per cent protein, 1.~4 per cent oil, 3.33 per cent fibre, 8.23 per cent ash, 4’04 per cent Ca, and 0.64 per cent P. The birds were maintained on these diets for a further 52 weeks and thus were 74 weeks old when killed. At the end of the period of feeding the birds were killed bv decaoitation and the rats bv cervical
dislocation. ‘Their‘ livers were rapidly iemoved, chilled, and portions homogenized with 4 volumes of ice-cold 0.1 M potassium phosphate buffer, #-I 7.0, containing 7.0 m&f 2-mercaptoethanol. The homogenates were centrifuged at xoo,ooog for I hour at o-4* C. (MSE supenpeed so), and the resulting supematant assayed for pyruvate kinase activity. The activity of pyruvate kinase (B.C. 2.7.1.40) was assayed by determining the
ht.
3. Biockmz.
rate of formation of pyruvate s~ctrophotome~itally in the presence of lactic dehydrogenase (B&her and Pfieiderer, 1955). Identical cuvettes were made up containing, in umoles: potassium phosphate buffer, PI-I 7.0, 200; M&I, IO; KC1 50; ADP 2.53 20 ~1. lactic dehydrogenase (equivalent 30 I.U.) ; and liver extract in a total volume of 2.7 ml. NADH (0.3 &mole) was added to the experimental cuvette and after the initial measurement of NADH dehydrogenase activity in the extract, Q fimoles phosphoenol pymvate were added and the decrease in absorption at 340 rng determined. The assays were carried out in a Unicam SP 800 recording spectrophotometer at 40” C. and in all cases the recorded activity was linear with respect to both time and protein
concentration in the extract. The protein content of cell-free extracts was determined by the biuret reaction (Layne, I957). RESULTS The results in Table I show the effects of feeding glucose and fructose for 4 days on the activity of hepatic pyruvate kinase (Experiment I) and it can be seen that there was no significant variation in the specific activity of the chick enzyme between dietary treatments. This experiment was also carried out using rats (Experiment 2) since in rats pyruvate kinase has been shown to vary in activity according to the type of carbohydrate in the diet. It was found that there was a difference between the specific activity in fructose-fed animals and in those receiving the other dietary treatments (Table I). Also given in Table I are some results from an earlier inv~tigation (Pearce, 1970) which similarly show that there was no significant variation of pyruvate kinase activity between treatments when diets containing 70 per cent fructose or 7o per cent glucose were fed to 6-week-old pullets for 4 days. Longer-term experiments were carried out in which the animals were on the experimental diets for 4 weeks (Table II). Again, no significant differences in the specific activities of chick pyruvate kinase were found between dietary treatments whereas in the rat the activity was greatest in the fructosefed animals (experiment 3 and 4). The effects of long-term fat feeding on hepatic pyruvate kinase activity in the adult laying hen (Experiment 5) are also shown in Table II. The results show that the dietary fat
50.6 fs.97 (4) rsg4.of 152.1 (6)
go per cent Fructose
46.7 rfr5.55 (5)* -
--
70 per cent Fructose 5r.29f5.87 673.1 fg4.9
60 per cent Fructose (4) (3)
47’43f7.07 548rt63.6
(4) (3)
80 per cent Glucose -_I____
DUET
(5)*
gr.34f7.r~ 1528.5 (2)
(6) g46of5*32 639-3 (2)
(6)
70 per cent Glucose g3.74f6.87 697.ofa4.r
(6) (3)
Cereal-based Control Diet
’
58.6f6.4 -
(3)
High Fat
Enzyme activities given as mpmoles per minute per mg. protein in the extract&S.E.M. of observations is given in parentheses.
-Chicken Rat
ANIMALS
RXET
(3)
The number
6a.4f3.6 -
Low Fat Control
Table I&--EFFECTSOF LONG-TERM FEEDING OFDIETSCONTASNING VARIOUSPROPORTIONS OFCARBOHYDRATE ORFAT ONTWESPECIFICACZT~V~TY OF HEPATK ~YRUVATEKINA~EINTHECHIC~CEN AND THE: RAT
EXPERIMENTAL
56.4r f7.or (4) 4g6*4f ~4’6 (6)
Control --
PYRUV~TE
is given in parentheses.
55’51 f5’2 1 (4) 468.4f49.2 (3)
60 per cent Glucose
The number olobservations
55’3f5.05 -
70 per cent Glucose
OF FEEDma DIETSCONTAINING DIFFERENTPNoeorwroNs OF GLWXE AND FRUCTOSEFOR 4 DAYS ON THE HEPATIC KINAS AortvfrY~~ THECH~CKEN ANDTHE RAT
Enzyme activities given as mumoles per minute per mg. protein in the extractfS.E.M. * These results taken from Pearce (1970).
Chicken Rat
EXPERIMENTAL ANIMAL
Table I.-EFFECTS
PEARCE
271
had no significant effect on the specific activity of this enzyme. It has been shown that long-term fat feeding significantly affects the activities of the lipogenic enzymes in the liver of the laying hen (Pearce, 1968) and this indicates that the non-adaptation of the pyruvate kinase is a true observation and is not an artefact due to the method of extract preparation. DISCUSSION
The results reported in this paper support 1970) that earlier observations (Pearce, pyruvate kinase activity in the domestic fowl is not significantly affected by diet. This observation was unexpected since pyruvate kinase is considered to be a ratelimiting glycolytic enzyme and has been shown to be susceptible to dietary manipulation. High-carbohydrate diets and particular diets containing fructose have been shown to increase the specific activity of hepatic pyruvate kinase in the rat (Krebs and Eggleston, 1965 ; Tanako, Harano, Sue, and Morimura, I 967 ; Bailey, Taylor, and Bartley, 1968; Szepesi and Freedland, 1g68a, b). In the present work the rat was used to obtain a comparison under the same experimental conditions and it was found that variation of pyruvate kinase activity between dietary treatments was readily detectable and showed the same pattern of activity as had been obtained by other workers. It is noteworthy that the specific activity of the enzyme was of the same order as that reported by Krebs and Eggleston (I 965) and Bailey, Taylor, and Bartley ( I 968). These results indicate that the techniques employed of tissue extract preparation and enzyme assay were capable of showing variation of enzyme specific activity in the rat which is known to exhibit the phenomenon and so the observed nonadaptation of pyruvate kinase activity in the domestic fowl was not an artefact. Further evidence that these results were not due to poor experimental technique was the observation that significant alterations of ketohexokinase activity were detected in the same extracts which show non-adaptation of pyruvate kinase (Pearce, 1970).
Dietary fat is also known to have an effect on pyruvate kinase activity in the rat. During the suckling period when the diet contains a high proportion of fat the specific activity of this enzyme is low compared with the postsuckling period and, similarly, placing rats from a low-fat diet on to one supplemented with fat results in a repression of pyruvate kinase activity (Taylor, Bailey, and Bartfey, I 967). The effect of dietary lipid on pyruvate kinase activity in the domestic fowl was exaciined in this experiment and again no variation in enzyme specific activity was found between dietary treatments. In contrast to the situation in the rat, mouse liver pyruvate kinase shows very little alteration in activity with changes in diet (Bailey, Taylor, and Bartley, 1968) and Gevers (1967) found that the enzyme in pigeon liver does not exhibit any variation in activity on starvation and refeeding. The situation in the domestic fowl would appear to be analogous to that in the pigeon and it seems probable that the activity of pyruvate kinase is not controlled at the level of enzymeprotein synthesis but at the level of interaction of the preformed enzyme with inhibitors (ATP, Cusf, alanine, proline, NADH, free fatty acids, ace+-CoA, and oxaloacetate) and activators (fructose- I ,6diphosphate, fructose- I -phosphate, glucose6-phosphate, and glyceraldehyde-S-phosphate) (Tanako and others, 1967 ; Taylor and Bailey, 1967 ; Bailey, Stirpe, and Taylor, 1968; Seubert, Henning, Schoner, and L’Age, 1968; Weber, Lea, and Stamm, 1968; Eggleston and Woods, 1970). Leveille ( 19%) has recently obtained evidence to show that pyruvate kinase from the liver of the chicken is allosterically activated by fructose-1,6diphosphate and this supports the view that regulation is effected at the level of preexisting enzyme. SUMiMARY I. The effect of dietary fructose, glucose, and lipid on hepatic pyruvate kinase activity in the domestic fowl was investigated. 2. Immature pullets were fed diets containing either 60, 70, or 80 per cent glucose or fructose or a cereal-based control diet for
‘97’,2
PYRUVATE KlNASEINDEPENDENT OF DIET
periods of 4 days and 4 weeks. Diets containing I 8 per cent dietary lipid or a cereal-based layers mash were fed to laying hens for 52 weeks 3. It was found that there was no significant variation in the specific activity of hepatic pyruvate kinase between dietary treatments. 4. The experiments were repeated using rats, and variation in specific activity of the enzyme between dietary treatments was readily detectable. 5. The results are discussed in relation to the mechanisms of control of pyruvate kinase which are known to operate in mammals. ACKNOWLEDGEMENTS The author wishes to thank the British Egg Marketing Board for the award of a Post-doctoral Research Fellowship, during the tenure of which part of this work was carried out. He also thanks Mr. E. Wallace for technical assistance. REFERENCES BAILEY,E., STIRPE,F., and TAYLOR,C. B. (1g68), ‘ Reaulation of rat-liver Dvruvate kinase. The effe; of preincubation pH,‘copper ions, fmctose diphosphate and dietary changes on enzyme activity ‘, Biochem. J., x08,417-436. BAILEY, I$ TAYLOR, C. B., and BARTLEY, W. (1968)9 Effect of dietary carbohydrate on hepatic lipogenesis in the rat ‘, Nature, Lond., 2x7,471-47= BBCWER,T., and PFL.EIDERER, G. (x955), ‘ Pyruvate kinase from muscle ‘, in Metfwds in Enrymology (ed. Co~ownx, S. P., and KAPLAN,N. a.), vol. I,,__ w. 4’35-640. *__ ._ New York : Academic Press. EGGLE~T~N,L. V., and Wools, H. F. (rg7o), ’ Activation of liver pyruvate kinase by fructcuex-phosphate ‘, FEB.9 L.etters, 6,43-45. FREEDLAXD,R. A., CUNLIFFE,T. L., and ZINKL, J. G. (1g66), ‘ The effect of insulin on enzyme adaptations to diets and hormones‘, J. bial. Chem., 41,54.48-545 I. GEVERB,W. (1g67), ‘ The regulation of phosphoenolpyruvate synthesis in pigeon liver ‘, Biachem. 39 103, 141-152. KREBS, H. A., and EGGLESTON, L. V. (1g65), ‘ The role of pyruvate kinase in the regulation of gluconeogenesis ‘, &o&m. j., w 3G4C. LAY% E. (1957), ’ Spectrophotometric and turbidimetric methods for measuring proteins.
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III. Biuret method ‘, in Methods in Enzpnolopl (ed. COLOWICK, S. P.,and KAPLAN,N.O.), vol. 3, pp. 450-451. New York: Academic Press. LEVEILLE.G. A. CI c&al. ‘ The influence of fructose diphosphatk &“pyruvate kinase activity in liver, muscle and adipose tissueof the rat, mouse, pig and chicken ‘, Camp. Biochem. Physiol., 28, 733-74o* PEARCE,J. (1g68), unpublished data. PEARCE,1. (Iqm), _. ., ‘ The effects of dietary fructose and ghxose on hepatic lipogenesis in the domestic fowl ‘. ht. 7. Biackm.. I. %06-st12. SEUBERT, W., HENNING:H. V., S&&R, W., and L’AGE, M. (1g68), ‘ Effects of cortisol on the levels of metabolites and enzymes controlling glucose production from pyruvate ‘, in Advances in Enzyme Regulation (ed. WEBER, G.), vol. 6, pp. I 53-180. London and New York : Pergamon. SZEPESI,B., and FREEDLAND,R. A. (1g68a), ’ Dietary regulation of pyruvate kinase synthesis in rat liver ‘, 3.JVutr., 9% 591-602. SZEPESI,B., and FREEDLAND,R. A. ( xg68b), ‘ Time course of enzyme adaptation. I. Effects of substituting dietary glucose and fructose at constant concentration of dietary protein ‘, Can. 3.B&x&m., 46, x45g-1470. TANAKO. T.. HARANO. Y., SUE. F., and MORIMURA,‘H.(I g67), ‘ C&&llization, ‘characterization and metabolic reguiation of two types of pyruvate kinase isolated from rat tissues ‘, 3. Bidsem., Taki, 6a, 71-91. TAYLOR,C. B., and BAILEY,E. (rg67), ‘ Activation of liver pyruvate kinase by fiustose-~$-diphosphate ‘, Biochem. J., 102, 32G33c. TAYLOR, C. B., BAILEY,E., and BARTLEY, W. ( Ig67), ‘ Changes in hepatic lipogenesis during development of the rat ‘, B&hem. J., 10%717722. WEBER,G., LEA, M. A., CONVERY, H. J. H., and STAMM,N. B. (1967), ‘ Regulation of gluconeogenesis and glycolysis; studies of mechanisms controlling enzyme activity ‘, in Adnames in Enryme apron (ed. WEBER, G.), vol. 5, London and New York: - 257-298. $Ergamon. WEBER, G., LEA, M. A., and ST-M, N. B. ( 1g68), ’ Sequential feedback inhibition and regulation of liver carbohydrate metabolism through control of enzyme activity ‘, in Advances in Enzpne Regulation (ed. W~BER, G.), vol. 6, pp101-123. London and New York: Pergamon. Kg Word Index: Dietary glucose, dietary fructose, dietary lipid, pyruvate kinase, rat, domestic fowl.