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Levels of some metabolites in liver of chick embryos and recently hatched chicks
LEVELS
OF SOME ~ETABOLIT~S IN LIVER CHICK EMBRYOS AND RECENTLY HATCHED CHICKS”
MARIA
TERESARINAUDO, CAROLA PONZETTO AND MAGDA
OF
CURIXI
Istituto di Chimica biologica dell’Universita’ di Torlno, Torino, Italy
Abstract-The following metabolites of the glycolytic pathway, along with ATP, ADP. AMP, citrate and ~uorganic phosphate, have been measured in the hepatic tissue of chick embryos and hatched chicks: glucose, ~u~os~-~-phosphate, fructose-~-phosphates fructose-1,6-diphosphate, dihydroxyacetone phosphate, ~ycer~debyde-3-phosphate, phosphoenol-pyruvate, pyruvate, and lactate.
data do not support this proposition since the enzyme shows absolute maximum activity between the 12th and, 14th days prior to hatching (Rinaudo, 1964). In order to elucidate the alternation of these basic pathways we have measured some compounds common to glycolysis and gluconeogenesis in chick embryo liver from the 8th day until hatching, and in the chick during the first few days after hatching. It is known that adenine nucleotides as well as other metabolites have a regulatory action upon pyruvate kinase, ~hospho~u~tok~n~e~ pyruvate carboxylaseandfructose-l,6-diphosphate-f-phosphohydrolase, which are key enzymes in glycolysis and gluconeogenesis. Measurements of the following compounds were taken from chick embryo liver on the 8th., 10th. 12th, l&h, 17th, 19th, and 21st days as well as a few days after hatching: glucose, glucose-ti-phosphate, fructose-6-phosphate, fructo~-1,4-diphosphate, dihydroxyacetone phosphate, ~ly~eraldehyde-3-~hosphate, phosphoenol pyruvate, pyruvate, lactate, citrate, inorganic phosphate, adenosine ~iphosphate, adenosine diphosphate and adenosine monophosphate.
INTRODUCTION
The hen’s egg contains a low level of free and combined carbohydrates, the concentration being around 0.1%. However the egg is rich in lipids and proteins from which the embryo may form its own carbohydrates, primarily glucose and glycogen. An increase in glycogen content is seen in the chick embryo liver between the 8th and 18th day (Rinaudo, 1961). This phenomenon is accompanied by a progressive increase of fructose-i,6”diphosphate-l-phosphoydrolase (Rinaudo, l%l), pyruvate carboxylase (Rinaudo & Ciunta, 19671and ~hosphoenol pyruvate carboxylase (Rinaudo, 1966). These enzymes are already active at 10 days and reach maximum peaks between 14 and 16 days, then slowly decrease. The activity maximum values are far greater than those in adults. Similarly the enzymes regulating the reversible steps of the glycolytic pathway show activity maximum values between the 14th and 18th day (Rinaudo, 1962, 1966). Between the 20th day and hatching, the level of glycogen drops abruptly. SimuI~neously phosphorylase and pyruvate kinase demonstrate a notable activation (Rinaudo, 1960, 1966). The resultant levels of activity are, however, far below adult levels. Based on the recorded data the following metabolic pattern might be proposed: gluconeogenesis and glycogen synthesis predominate up until the 17-18th day in chick embryo hepatic tissue. During this period the glycolytic pathway would be expected to show low activity. In the succeeding days until hatching the glycolytic pathway should show an increase in activity becoming predominately active a few days after hatching. The referenced phosphofru~tokinase
*Work supported by a grant from the C.N.R. (National Research Council).
MATERIALS
AND
iMETHODS
Eggs from Leghorn hens were used for the experiments. Incubation was performed at 37°C and humidity saturation. Embryos were immediately separated from the extraembryonic membranes and their livers removed as quickly as possible. The livers were then immediately immersed in liquid nitrogen until use. The hepatic tissue was then crushed in a mortar while continuously adding liquid nitrogen, HQO, 6% was added and the tissue homogenized at low temperature. Centrifuging followed in a refrigerated Spinco at 100,~~ for 30min in ZXZCUU~. The supe~natant &as then brought to pH 5.6-6 using 10 N KOH and again centrifuged at 30,OOOg for 10 mm to separate the insoluble KCiU,.
239
MARIA TEKESA RINALIDO. CAHOLA POSZFTTO AND MAC;DA Ct xx)
240
Metaholite determrnatlon were made by spectrophotometric methods. A Beckman DK-2A spectrophotometer with 4 cm cells was used to measure G-6-P (Hohorst, 1970) F-6-P (Hohorst. 1970) FDP (Bucher & Hohorst, 1970) DAP (Bucher & Hohorst, 1970), GAP (Bucher & Hohorst. 1970). PEP (Czock & Lamprecht, 1970). pyruvate (Czock & Lamprecht. 1970) and citrate (Dagley. 1970) at 340 nm. An Eppendorf photometer with 1 cm cells was used to measure glucose (Bergmeyer et ai., 1970) lactate (Hohorst, 1970), ATP (Lamprecht & Trantschold. 1970) and AMP (Jaworek et al.. 1970) at 366 nm. Inorganic phosphate was measured by calorimetry. according to Youngburg & Youngburg (1930). The extract was prepared in lO”d TCA using a procedure similar to the previously mentroncd preparation in HClO,. For each metabolite the figures show maximum and minimum oscillations from the median. These values are the results of 48 measurements, each one utilizing Z(fl4 embryos for the period between 8 and 14 day respectively. For the period following the 14th day until hatching, IO-6 embryos were used; while at 36 hr and 12 days after hatching. 4 embryos were used for each measurement.
RESULTS
1 shows glucose
and
150130IO go705030-
levels in the liver of chick embryo and hatched chick. Glucose demonstrates continual accumulation during the embryonal development. Lactic acid remains constant between the 10th and 12th day, drops successively and rises after the 17th day until hatching. The Figure
B
lactic
acid
IOI
8
,
10
I,, 8
*3
12
,
,
,
14
16
%
,
I
----
hatchlng36h
12
days
Fig. I. Glucose
and lactic acid levels in liver of the deve-
loping chrck embryo and hatched chick.
t
9
14
16
.
18
m--_-7,
ho&g
36f,
12
day5
Fig. 2. G-6-P, F-6-P, and FDP levels in liver of the developing
10‘6MOia5/g tG%e
/
12
chick embryo
and hatched
chick.
measurements in the hatched chick showed increased levels for both compounds far above embryonal levels. Figure 2 shows levels of G-6-P. F-6-P and FDP in the liver of chick embryo and hatched chick. G-6-P shows a continuous accumulation with a maximum on the 17th day, after which it drops until hatching. F-6-P and FDP maintain more or less constant levels until hatching. All three compounds markedly increase on the 12th day after hatching, G-6-P increase being more accentuated. Measured levels for DAP, GAP. PEP and pyruvate are represented in Fig. 3. DAP and GAP show hmited oscillations until hatching and reach m~imum concentrations on the 12th day after hatching. The highest embryonal concentrations of PEP and pyruvate were found on the 8th day. Pyruvate drops markedly until the 17th day and levels off. PEP concentration diminishes until the 16th day after which it rises to a new maximum on the 12th day after hatching. The graphs of Fig. 4 show ATP. ADP and AMP concentrations measured in liver of chick embryo and hatched chick. ATP demonstrates a continuous fall in concentration during the embryonal and posthatching period. The final concentration on the 12th day after hatching is one fourth that at 8 days of embryonal Iife. Prenatal and postnatal levels for ADP
241
Metabolites in liver of chicks 166MoleS/9
10~sMolos/gtissue
tlssua
100
1.60.
25-
15-
0.60
=1
‘------
12
14
16
18
hatching
36h
-
12
days
Fig. 4. ATP, ADP and AMP levels in liver of the developing chick embryo and hatched chick.
II
0
10
_____.--r,
I
*
e
I
12
14
16
18
II
hatching
’
36h
10d Moles/g
12
days
Fig. 3. DAP, GAP, PEP and pyruvate levels in liver of the developing chick embryo and hatched chick. 6
I 1
are not greatly varied. The embryonal ADP levels at the 12-14th day are almost identical to those at the 12th postnatal day. AMP demonstrates a gradual increase which is accentuated just after hatching, hence the postnatal concentrations are markedly superior to prenatal concentrations. Figure 5 represents embryonal and post-hatching levels for inorganic phosphate and citrate. An analogy between citrate and ATP curves may be noted, in as much as both compounds show maxima at 8 days prenatal which are higher than levels found during the remaining prenatal-postnatal period. Inorganic phosphate increases steadily between the 8th and 14th day, reaches a peak between days 14 and 17, then decreases. Table 1 shows relationships of the measured adenine nucleotides: ATP/AMP and ATP1ADP.P. Equilibrium constants for the reactions catalized by phosphofructokinase and pyruvate kinase, calculated using the median levels of ATP, ADP, FDP, F-6-P, PEP and pyruvate, are also in Table 1. Dephosphorylated forms of these nucleotides have an ever-increasing concentration during the course of
;
tbssue
nnorgamf phosphate
/my+-_
(-----
14
16
18
hatching
36h
12
dilYS
Fig. 5. Inorganic phosphate and citrate levels in liver of the developing chick embryo and hatched chick.
242 Table
Tt w SA RINAI~IX~. CAKOLA PONZLTTO A\I) MAW),\ C‘I RIO
MAW I. Relationships
between
[ATP] -[AMP]. kinase
[ATP];[AMP] Embryo 8 days Embryo IO days Embryo 12 days Embryo 14 days Embryo 17 days Embryo 19 days Hatching Chick 36 hr Chick 12 days
I .4X6 I.817 I.376 I. I77 0.777 0.7 I9 0.489 0.3 I7 0.240
[ATP] [ADP]. [P] and equilibrium and pyruvate kinase reactions
[ATP]JADP] 0.3% 0.48’) 0.253 0.243 0.1 x5 0.203 0.179 0. I24 0.118
The equilibrium constants for the two kinase-catalized reactions differ notably from those calculated from in vitro reactions (Burton & Krebs, 1953). These constants support previously obtained data indicating that phosphofructokinase in chick embryo liver acquires maximum activity between the 10 and 14th prenatal day. superior to adult activity levels. Pyruvate kinase, on the contrary. shows lowest levels of activity in the initial embryonal period. rising constantly after the 17th day. development.
DISCUSSION
The results seem to confirm that hepatic glycogen, which increases progressively during the embryonal development, reaching a maximum on the 17.- 18th day, originates by gluconeogenesis from noncarbohydrate precursors. In fact pyruvate. with a maximum at 8 embryonal days. lowers constantly, leveling around the 17-18th day. Lactic acid, constant in the 8-12 day interval, lowers thereafter until the 17th day. In this 8-17 day time period the catalytic capacity of the gluconeogenesis regulatory enzymes is at a maximum while the phosphorylase and pyruvate kinase activities are extremely low. G-6-P increases until the 17th day, after which it rapidly drops: this fall may be explained by the conspicuous increase not only of hepatic glycogen but also of hepatic glucose (Yarnell rf uI., 1966). In accordance with this data the glucose-6-phosphatase has been found to be markedly active during the same embryonal period (Rinaudo. 1960). After the 18th day a metabolic situation favorable to glycolytic flow is indicated by the phosphorylase and pyruvate kinase activation, pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase inactivation, and confirmed by a net increase in lactate with lowering of glycogen levels. The regulation of glycolysis and gluconeogenesis may be explained by data in Figs. 4 and 5 and Table I, particularly significant being the ratios ATP/AMP and ATP/ADP.P. In the embryo liver ATP and citrate are in maximum concentration on the 8th day.
[P]
constants
[FDP].[ADP], [F6P]. [ATP]
01 the phosphofructo-
[Pyr] [ATP]/ [PEP]. [ADP]
0.603 0.337 0.406 0.3x9 0.455 O.SOY 0.575 I.337 1.874
4.32 4.21 3.40 4.13 3.01 1.7’ I .07 0.55 0. IY
AMP increases continuously during embryonal development and reaches its maximum value after hatching. ATP and citrate elevated levels repress phosphofructokinase while ADP and AMP activate this enzyme. ATP is a pyruvate carboxylase positive modulator and AMP is a fructose diphosphatase negative modulator. The ATP/ADP ratio plus the AMP and citrate concentration. are favorable to activation of the gluconeogenesis enzymes in the precocious embryonal period. After the 17th day the relationship among the adenine nucleotides, and citrate concentration become favorable to an activation of glycolysis enzymes. This alternation of metabolic pathways is further confirmed by the measured levels of inorganic phosphate, which increase until the 17th day and then lower. An increase in inorganic phosphate pushes to the left the reaction catalized by glyceraldehyde-3phosphate dehydrogenase: [!%?I_
= k ._!X!:PGA!
CNADH,I
[GAPI. [PiI
The resulting increase in NADH,, deduced from simultaneous diminished lactate levels, is thought to favor the transformation of 1,3-diphosphoglycerate into glyceraldehyde-3-phosphate and to finally favor glucose synthesis until the 17th day. The opposite situation prevails after the 17th day with lowered inorganic phosphate and thus indirectly NADH,, favoring glycolysis. ATP, ADP, AMP and Pi have shown analogous variation in livers of rats under condition of starvation or high-fat diet, both conditions rendering a gluconeogenesis activation (Krebs, 1971). The measured activity of phosphofructokinase as well as the equilibrium constant calculated from our data do not readily fit into the metabolic picture described up to this point. This enzyme shows maximum activity during the embryonal period which is otherwise indicative of gluconeogenesis. In the hatched chick phosphofructokinase activity is diminished while the other glycolytic enzyme activities
Metabolites in liver of chicks are increased. Recent observations by Kono et al. (1974) may help to explain this apparent contradiction. These authors have used crude as well as purified preparations of phosphofructokinase and fructose diphosphatase from chick liver. They demonstrated that phosphofructokinase can bind with fructose diphosphatase to form a complex of varying sensibility to ATP, according to the number of fructose diphosphatase molecules in bound condition. If this number is high the kinase is more sensitive to ATP, while if this number is low the sensitivity is negligible. The phosphofructokinase in chick embryo liver, by complexing with fructose diphosphatase, may hence exist in a form less sensitive to ATP, which permits it to function even in the presence of ATP. Glycolysis in chick embryo liver does not appear to be controlled by phosphofructokinase but rather by phosphorylase and especially by pyruvate kinase. A controlling influence on this last enzyme is exercized by ATP, ADP and AMP as well as pyruvate. SUMMARY
The following metabolites of the glycolytic pathway, along with ATP, ADP, AMP, citrate and inorganic phosphate have been measured in the hepatic tissue of chick embryos and hatched chicks: glucose, G-6-P, F-6-P, FDP, DAP, GAP, PEP, pyruvate and lactate. 1. Glucose shows an a~umulation during the entire embryonal development, while lactate is at constant levels at 8-12 days, lowers from 14-17 days, and rises in successive days. 2. G-6-P reaches maximum concentration on the 17th day and then reduces until hatching. 3. FDP and F-6-P show a slight but constant increase during the entire embryonal period. Glucose, G-6-P, F-6-P and FDP in the hatched chick showed levels superior to those in the embryo. 4. DAP and GAP show limited oscillations until hatching 5. Pyruvate has a maximum embryonal level on the 8th day after which it falls rather rapidly to the 18th day and successively descends to a minimum on the 12th day after hatching. PEP also has an embryonal maximum level on the 8th day, however falls to a minimum near to the 16th day and successively rises to an absolute maximum on the 12th day after hatching. 6. ATP demonstrates a continuous fall in concentration during the embryonal and post-hatching period. Prenatal and postnatal levels for ADP are not greatly varied. AMP demonstrates a gradual increase, which is accentuated after hatching. 7. The citrate pattern is similar to that of ATP. 8. Inorganic phosphate accumulates to a maximum level at 17 days, roughly 30% higher than adult levels. REFERENCES
BERGMEYER H. U., BERNT E., SCHMIDTF. & SXORK H. (1970) D-glucose: Bestimmung mit Hexokinase und Glu-
243
case-6-phosphat-Dehydrogenase in ~ethoden der enzy~fisc~e~ Analyse (Edited by BERG~MEYER H. U.) Vol. 2, p. 1162.Verlag Chemie, Weinheim. BUCHERT. & HOHORSTH. J. (1970) D-Fructose-1,6-diphosphat, Dihydrox~aceton-phosphat und Glycerinaldehyd-3-phosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYEK H. U.) Vol. 2, p. 1282. Verlag Chemie, Weinheim. BURTONK. & KREBSH. A. (1953) The free-energy changes associated with the individual steps of the tricarboxylic acid cycle, glycolysis and alcoholic fermentation and with the hydrolysis of the pyrophosphate groups of adenosinetriphosphate. Biochem. J. 54, 94107. CZOCKR. & LAMPRECH~ W. (1970) Pyruvat, Phosphoenolpyruvat und D-Glycerat-2-phosphat in Methoden der enzymutjsche~ Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1407. Verlag Chemie, Weinheim. DAGLEYS. (1970) Citrat: u.v.-spektrophotometrische Bestimmung in ~et~iode~ der e~zymutischeri Anatpr (Edited by BERGMEYER H. U,) Vol. 2, p. 1520. Verlag Chemie. Weinheim, HOHORSTH. J. (1970) DGlucose-6-phosphat und D-Fructose-6-phosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1201. Verlag Chemie, Weinheim. HOHORSTH. J. (1970) L-(+ )-Lactat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1425. Verlag Chemie, Weinheim. JAWOREKD., GRUBER W. & BERGMEYER H. U. (1970) Adenosin-S-diphosphat und Adenosin-S-monophosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 205. Verlag Chemie, Weinheim. KONO N. & UYEDAK. (1974) Chicken liver phosphofructokinase--III. Kinetics and allosteric properties. 1. biol. Chem. 249. 1490-1496.
KREBSH. A. (1971) Gluconeogenesis and Redox State in Regulation of GIuconeogenesis (Edited by SOLINGH. D. & WILLMSB.) pp. 114-117. Academic Press, NY. LAMPRECHT W. & TRANTXHOLDI. (1970) Adenosin-5’-triphosphat: Bestimmung mit Hexokinase und Glucose-6phosphat Dehydrogenase in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p, 2024. Verlag Chemie, Weinheim. RINAUW M. T. (1960) Glucose-6-fosfatasi nel fegato di embrione di Callus Domesricus. Arch. Sci. Biol. 44, 346-349.
RINAUW M. T. (1960) Fosforilasi e fosfoglu~omutasi nel fegato di embrione di Gallus ~or~les~~c~s. Boll. Sot. It. Biol. Sper. 36. 2004-2007. RINAUM) M. T. (1961) Fructose-t,&dephosphatase in the liver of chickens’ embryos. Exprrientia 17. 3C-31. RINAUW M. T. (1962) Glycogenesis in the liver of chicken embryo. Enzymologia 24, 230-236. RINAUDOM. T. (1966) Enzymes of glycolysis and glycogenesis in the liver of chicken embryo. Enzymologia 31. 325-333.
RINAUDOM. T. & GIUNTA C. (1967) Pyruvate carboxylase, malate dehydrogenase, fumarase and aspartate aminotransferase in the mitochondria and in the cytoplasm of liver of chicken embryo. Enzymologia 33, 201-210. YARNELL,G. R., NELSONP. A. & WAGLES. R. (1966) Biochemical studies of the developing embryo-I. Metabolism of gluconeogenic precursors. Arch. B~chem. Biophw. 114, 539-542. YOUNGBURG G. E. & YOUNGBURG M. V.(1930) A system of blood phosphorus anaiysis. J. lab. Clin. 16, 158-166.
OF SOME ~ETABOLIT~S IN LIVER CHICK EMBRYOS AND RECENTLY HATCHED CHICKS”
MARIA
TERESARINAUDO, CAROLA PONZETTO AND MAGDA
OF
CURIXI
Istituto di Chimica biologica dell’Universita’ di Torlno, Torino, Italy
Abstract-The following metabolites of the glycolytic pathway, along with ATP, ADP. AMP, citrate and ~uorganic phosphate, have been measured in the hepatic tissue of chick embryos and hatched chicks: glucose, ~u~os~-~-phosphate, fructose-~-phosphates fructose-1,6-diphosphate, dihydroxyacetone phosphate, ~ycer~debyde-3-phosphate, phosphoenol-pyruvate, pyruvate, and lactate.
data do not support this proposition since the enzyme shows absolute maximum activity between the 12th and, 14th days prior to hatching (Rinaudo, 1964). In order to elucidate the alternation of these basic pathways we have measured some compounds common to glycolysis and gluconeogenesis in chick embryo liver from the 8th day until hatching, and in the chick during the first few days after hatching. It is known that adenine nucleotides as well as other metabolites have a regulatory action upon pyruvate kinase, ~hospho~u~tok~n~e~ pyruvate carboxylaseandfructose-l,6-diphosphate-f-phosphohydrolase, which are key enzymes in glycolysis and gluconeogenesis. Measurements of the following compounds were taken from chick embryo liver on the 8th., 10th. 12th, l&h, 17th, 19th, and 21st days as well as a few days after hatching: glucose, glucose-ti-phosphate, fructose-6-phosphate, fructo~-1,4-diphosphate, dihydroxyacetone phosphate, ~ly~eraldehyde-3-~hosphate, phosphoenol pyruvate, pyruvate, lactate, citrate, inorganic phosphate, adenosine ~iphosphate, adenosine diphosphate and adenosine monophosphate.
INTRODUCTION
The hen’s egg contains a low level of free and combined carbohydrates, the concentration being around 0.1%. However the egg is rich in lipids and proteins from which the embryo may form its own carbohydrates, primarily glucose and glycogen. An increase in glycogen content is seen in the chick embryo liver between the 8th and 18th day (Rinaudo, 1961). This phenomenon is accompanied by a progressive increase of fructose-i,6”diphosphate-l-phosphoydrolase (Rinaudo, l%l), pyruvate carboxylase (Rinaudo & Ciunta, 19671and ~hosphoenol pyruvate carboxylase (Rinaudo, 1966). These enzymes are already active at 10 days and reach maximum peaks between 14 and 16 days, then slowly decrease. The activity maximum values are far greater than those in adults. Similarly the enzymes regulating the reversible steps of the glycolytic pathway show activity maximum values between the 14th and 18th day (Rinaudo, 1962, 1966). Between the 20th day and hatching, the level of glycogen drops abruptly. SimuI~neously phosphorylase and pyruvate kinase demonstrate a notable activation (Rinaudo, 1960, 1966). The resultant levels of activity are, however, far below adult levels. Based on the recorded data the following metabolic pattern might be proposed: gluconeogenesis and glycogen synthesis predominate up until the 17-18th day in chick embryo hepatic tissue. During this period the glycolytic pathway would be expected to show low activity. In the succeeding days until hatching the glycolytic pathway should show an increase in activity becoming predominately active a few days after hatching. The referenced phosphofru~tokinase
*Work supported by a grant from the C.N.R. (National Research Council).
MATERIALS
AND
iMETHODS
Eggs from Leghorn hens were used for the experiments. Incubation was performed at 37°C and humidity saturation. Embryos were immediately separated from the extraembryonic membranes and their livers removed as quickly as possible. The livers were then immediately immersed in liquid nitrogen until use. The hepatic tissue was then crushed in a mortar while continuously adding liquid nitrogen, HQO, 6% was added and the tissue homogenized at low temperature. Centrifuging followed in a refrigerated Spinco at 100,~~ for 30min in ZXZCUU~. The supe~natant &as then brought to pH 5.6-6 using 10 N KOH and again centrifuged at 30,OOOg for 10 mm to separate the insoluble KCiU,.
239
MARIA TEKESA RINALIDO. CAHOLA POSZFTTO AND MAC;DA Ct xx)
240
Metaholite determrnatlon were made by spectrophotometric methods. A Beckman DK-2A spectrophotometer with 4 cm cells was used to measure G-6-P (Hohorst, 1970) F-6-P (Hohorst. 1970) FDP (Bucher & Hohorst, 1970) DAP (Bucher & Hohorst, 1970), GAP (Bucher & Hohorst. 1970). PEP (Czock & Lamprecht, 1970). pyruvate (Czock & Lamprecht. 1970) and citrate (Dagley. 1970) at 340 nm. An Eppendorf photometer with 1 cm cells was used to measure glucose (Bergmeyer et ai., 1970) lactate (Hohorst, 1970), ATP (Lamprecht & Trantschold. 1970) and AMP (Jaworek et al.. 1970) at 366 nm. Inorganic phosphate was measured by calorimetry. according to Youngburg & Youngburg (1930). The extract was prepared in lO”d TCA using a procedure similar to the previously mentroncd preparation in HClO,. For each metabolite the figures show maximum and minimum oscillations from the median. These values are the results of 48 measurements, each one utilizing Z(fl4 embryos for the period between 8 and 14 day respectively. For the period following the 14th day until hatching, IO-6 embryos were used; while at 36 hr and 12 days after hatching. 4 embryos were used for each measurement.
RESULTS
1 shows glucose
and
150130IO go705030-
levels in the liver of chick embryo and hatched chick. Glucose demonstrates continual accumulation during the embryonal development. Lactic acid remains constant between the 10th and 12th day, drops successively and rises after the 17th day until hatching. The Figure
B
lactic
acid
IOI
8
,
10
I,, 8
*3
12
,
,
,
14
16
%
,
I
----
hatchlng36h
12
days
Fig. I. Glucose
and lactic acid levels in liver of the deve-
loping chrck embryo and hatched chick.
t
9
14
16
.
18
m--_-7,
ho&g
36f,
12
day5
Fig. 2. G-6-P, F-6-P, and FDP levels in liver of the developing
10‘6MOia5/g tG%e
/
12
chick embryo
and hatched
chick.
measurements in the hatched chick showed increased levels for both compounds far above embryonal levels. Figure 2 shows levels of G-6-P. F-6-P and FDP in the liver of chick embryo and hatched chick. G-6-P shows a continuous accumulation with a maximum on the 17th day, after which it drops until hatching. F-6-P and FDP maintain more or less constant levels until hatching. All three compounds markedly increase on the 12th day after hatching, G-6-P increase being more accentuated. Measured levels for DAP, GAP. PEP and pyruvate are represented in Fig. 3. DAP and GAP show hmited oscillations until hatching and reach m~imum concentrations on the 12th day after hatching. The highest embryonal concentrations of PEP and pyruvate were found on the 8th day. Pyruvate drops markedly until the 17th day and levels off. PEP concentration diminishes until the 16th day after which it rises to a new maximum on the 12th day after hatching. The graphs of Fig. 4 show ATP. ADP and AMP concentrations measured in liver of chick embryo and hatched chick. ATP demonstrates a continuous fall in concentration during the embryonal and posthatching period. The final concentration on the 12th day after hatching is one fourth that at 8 days of embryonal Iife. Prenatal and postnatal levels for ADP
241
Metabolites in liver of chicks 166MoleS/9
10~sMolos/gtissue
tlssua
100
1.60.
25-
15-
0.60
=1
‘------
12
14
16
18
hatching
36h
-
12
days
Fig. 4. ATP, ADP and AMP levels in liver of the developing chick embryo and hatched chick.
II
0
10
_____.--r,
I
*
e
I
12
14
16
18
II
hatching
’
36h
10d Moles/g
12
days
Fig. 3. DAP, GAP, PEP and pyruvate levels in liver of the developing chick embryo and hatched chick. 6
I 1
are not greatly varied. The embryonal ADP levels at the 12-14th day are almost identical to those at the 12th postnatal day. AMP demonstrates a gradual increase which is accentuated just after hatching, hence the postnatal concentrations are markedly superior to prenatal concentrations. Figure 5 represents embryonal and post-hatching levels for inorganic phosphate and citrate. An analogy between citrate and ATP curves may be noted, in as much as both compounds show maxima at 8 days prenatal which are higher than levels found during the remaining prenatal-postnatal period. Inorganic phosphate increases steadily between the 8th and 14th day, reaches a peak between days 14 and 17, then decreases. Table 1 shows relationships of the measured adenine nucleotides: ATP/AMP and ATP1ADP.P. Equilibrium constants for the reactions catalized by phosphofructokinase and pyruvate kinase, calculated using the median levels of ATP, ADP, FDP, F-6-P, PEP and pyruvate, are also in Table 1. Dephosphorylated forms of these nucleotides have an ever-increasing concentration during the course of
;
tbssue
nnorgamf phosphate
/my+-_
(-----
14
16
18
hatching
36h
12
dilYS
Fig. 5. Inorganic phosphate and citrate levels in liver of the developing chick embryo and hatched chick.
242 Table
Tt w SA RINAI~IX~. CAKOLA PONZLTTO A\I) MAW),\ C‘I RIO
MAW I. Relationships
between
[ATP] -[AMP]. kinase
[ATP];[AMP] Embryo 8 days Embryo IO days Embryo 12 days Embryo 14 days Embryo 17 days Embryo 19 days Hatching Chick 36 hr Chick 12 days
I .4X6 I.817 I.376 I. I77 0.777 0.7 I9 0.489 0.3 I7 0.240
[ATP] [ADP]. [P] and equilibrium and pyruvate kinase reactions
[ATP]JADP] 0.3% 0.48’) 0.253 0.243 0.1 x5 0.203 0.179 0. I24 0.118
The equilibrium constants for the two kinase-catalized reactions differ notably from those calculated from in vitro reactions (Burton & Krebs, 1953). These constants support previously obtained data indicating that phosphofructokinase in chick embryo liver acquires maximum activity between the 10 and 14th prenatal day. superior to adult activity levels. Pyruvate kinase, on the contrary. shows lowest levels of activity in the initial embryonal period. rising constantly after the 17th day. development.
DISCUSSION
The results seem to confirm that hepatic glycogen, which increases progressively during the embryonal development, reaching a maximum on the 17.- 18th day, originates by gluconeogenesis from noncarbohydrate precursors. In fact pyruvate. with a maximum at 8 embryonal days. lowers constantly, leveling around the 17-18th day. Lactic acid, constant in the 8-12 day interval, lowers thereafter until the 17th day. In this 8-17 day time period the catalytic capacity of the gluconeogenesis regulatory enzymes is at a maximum while the phosphorylase and pyruvate kinase activities are extremely low. G-6-P increases until the 17th day, after which it rapidly drops: this fall may be explained by the conspicuous increase not only of hepatic glycogen but also of hepatic glucose (Yarnell rf uI., 1966). In accordance with this data the glucose-6-phosphatase has been found to be markedly active during the same embryonal period (Rinaudo. 1960). After the 18th day a metabolic situation favorable to glycolytic flow is indicated by the phosphorylase and pyruvate kinase activation, pyruvate carboxylase, phosphoenolpyruvate carboxylase and fructose diphosphatase inactivation, and confirmed by a net increase in lactate with lowering of glycogen levels. The regulation of glycolysis and gluconeogenesis may be explained by data in Figs. 4 and 5 and Table I, particularly significant being the ratios ATP/AMP and ATP/ADP.P. In the embryo liver ATP and citrate are in maximum concentration on the 8th day.
[P]
constants
[FDP].[ADP], [F6P]. [ATP]
01 the phosphofructo-
[Pyr] [ATP]/ [PEP]. [ADP]
0.603 0.337 0.406 0.3x9 0.455 O.SOY 0.575 I.337 1.874
4.32 4.21 3.40 4.13 3.01 1.7’ I .07 0.55 0. IY
AMP increases continuously during embryonal development and reaches its maximum value after hatching. ATP and citrate elevated levels repress phosphofructokinase while ADP and AMP activate this enzyme. ATP is a pyruvate carboxylase positive modulator and AMP is a fructose diphosphatase negative modulator. The ATP/ADP ratio plus the AMP and citrate concentration. are favorable to activation of the gluconeogenesis enzymes in the precocious embryonal period. After the 17th day the relationship among the adenine nucleotides, and citrate concentration become favorable to an activation of glycolysis enzymes. This alternation of metabolic pathways is further confirmed by the measured levels of inorganic phosphate, which increase until the 17th day and then lower. An increase in inorganic phosphate pushes to the left the reaction catalized by glyceraldehyde-3phosphate dehydrogenase: [!%?I_
= k ._!X!:PGA!
CNADH,I
[GAPI. [PiI
The resulting increase in NADH,, deduced from simultaneous diminished lactate levels, is thought to favor the transformation of 1,3-diphosphoglycerate into glyceraldehyde-3-phosphate and to finally favor glucose synthesis until the 17th day. The opposite situation prevails after the 17th day with lowered inorganic phosphate and thus indirectly NADH,, favoring glycolysis. ATP, ADP, AMP and Pi have shown analogous variation in livers of rats under condition of starvation or high-fat diet, both conditions rendering a gluconeogenesis activation (Krebs, 1971). The measured activity of phosphofructokinase as well as the equilibrium constant calculated from our data do not readily fit into the metabolic picture described up to this point. This enzyme shows maximum activity during the embryonal period which is otherwise indicative of gluconeogenesis. In the hatched chick phosphofructokinase activity is diminished while the other glycolytic enzyme activities
Metabolites in liver of chicks are increased. Recent observations by Kono et al. (1974) may help to explain this apparent contradiction. These authors have used crude as well as purified preparations of phosphofructokinase and fructose diphosphatase from chick liver. They demonstrated that phosphofructokinase can bind with fructose diphosphatase to form a complex of varying sensibility to ATP, according to the number of fructose diphosphatase molecules in bound condition. If this number is high the kinase is more sensitive to ATP, while if this number is low the sensitivity is negligible. The phosphofructokinase in chick embryo liver, by complexing with fructose diphosphatase, may hence exist in a form less sensitive to ATP, which permits it to function even in the presence of ATP. Glycolysis in chick embryo liver does not appear to be controlled by phosphofructokinase but rather by phosphorylase and especially by pyruvate kinase. A controlling influence on this last enzyme is exercized by ATP, ADP and AMP as well as pyruvate. SUMMARY
The following metabolites of the glycolytic pathway, along with ATP, ADP, AMP, citrate and inorganic phosphate have been measured in the hepatic tissue of chick embryos and hatched chicks: glucose, G-6-P, F-6-P, FDP, DAP, GAP, PEP, pyruvate and lactate. 1. Glucose shows an a~umulation during the entire embryonal development, while lactate is at constant levels at 8-12 days, lowers from 14-17 days, and rises in successive days. 2. G-6-P reaches maximum concentration on the 17th day and then reduces until hatching. 3. FDP and F-6-P show a slight but constant increase during the entire embryonal period. Glucose, G-6-P, F-6-P and FDP in the hatched chick showed levels superior to those in the embryo. 4. DAP and GAP show limited oscillations until hatching 5. Pyruvate has a maximum embryonal level on the 8th day after which it falls rather rapidly to the 18th day and successively descends to a minimum on the 12th day after hatching. PEP also has an embryonal maximum level on the 8th day, however falls to a minimum near to the 16th day and successively rises to an absolute maximum on the 12th day after hatching. 6. ATP demonstrates a continuous fall in concentration during the embryonal and post-hatching period. Prenatal and postnatal levels for ADP are not greatly varied. AMP demonstrates a gradual increase, which is accentuated after hatching. 7. The citrate pattern is similar to that of ATP. 8. Inorganic phosphate accumulates to a maximum level at 17 days, roughly 30% higher than adult levels. REFERENCES
BERGMEYER H. U., BERNT E., SCHMIDTF. & SXORK H. (1970) D-glucose: Bestimmung mit Hexokinase und Glu-
243
case-6-phosphat-Dehydrogenase in ~ethoden der enzy~fisc~e~ Analyse (Edited by BERG~MEYER H. U.) Vol. 2, p. 1162.Verlag Chemie, Weinheim. BUCHERT. & HOHORSTH. J. (1970) D-Fructose-1,6-diphosphat, Dihydrox~aceton-phosphat und Glycerinaldehyd-3-phosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYEK H. U.) Vol. 2, p. 1282. Verlag Chemie, Weinheim. BURTONK. & KREBSH. A. (1953) The free-energy changes associated with the individual steps of the tricarboxylic acid cycle, glycolysis and alcoholic fermentation and with the hydrolysis of the pyrophosphate groups of adenosinetriphosphate. Biochem. J. 54, 94107. CZOCKR. & LAMPRECH~ W. (1970) Pyruvat, Phosphoenolpyruvat und D-Glycerat-2-phosphat in Methoden der enzymutjsche~ Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1407. Verlag Chemie, Weinheim. DAGLEYS. (1970) Citrat: u.v.-spektrophotometrische Bestimmung in ~et~iode~ der e~zymutischeri Anatpr (Edited by BERGMEYER H. U,) Vol. 2, p. 1520. Verlag Chemie. Weinheim, HOHORSTH. J. (1970) DGlucose-6-phosphat und D-Fructose-6-phosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1201. Verlag Chemie, Weinheim. HOHORSTH. J. (1970) L-(+ )-Lactat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 1425. Verlag Chemie, Weinheim. JAWOREKD., GRUBER W. & BERGMEYER H. U. (1970) Adenosin-S-diphosphat und Adenosin-S-monophosphat in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p. 205. Verlag Chemie, Weinheim. KONO N. & UYEDAK. (1974) Chicken liver phosphofructokinase--III. Kinetics and allosteric properties. 1. biol. Chem. 249. 1490-1496.
KREBSH. A. (1971) Gluconeogenesis and Redox State in Regulation of GIuconeogenesis (Edited by SOLINGH. D. & WILLMSB.) pp. 114-117. Academic Press, NY. LAMPRECHT W. & TRANTXHOLDI. (1970) Adenosin-5’-triphosphat: Bestimmung mit Hexokinase und Glucose-6phosphat Dehydrogenase in Methoden der enzymatischen Analyse (Edited by BERGMEYER H. U.) Vol. 2, p, 2024. Verlag Chemie, Weinheim. RINAUW M. T. (1960) Glucose-6-fosfatasi nel fegato di embrione di Callus Domesricus. Arch. Sci. Biol. 44, 346-349.
RINAUW M. T. (1960) Fosforilasi e fosfoglu~omutasi nel fegato di embrione di Gallus ~or~les~~c~s. Boll. Sot. It. Biol. Sper. 36. 2004-2007. RINAUM) M. T. (1961) Fructose-t,&dephosphatase in the liver of chickens’ embryos. Exprrientia 17. 3C-31. RINAUW M. T. (1962) Glycogenesis in the liver of chicken embryo. Enzymologia 24, 230-236. RINAUDOM. T. (1966) Enzymes of glycolysis and glycogenesis in the liver of chicken embryo. Enzymologia 31. 325-333.
RINAUDOM. T. & GIUNTA C. (1967) Pyruvate carboxylase, malate dehydrogenase, fumarase and aspartate aminotransferase in the mitochondria and in the cytoplasm of liver of chicken embryo. Enzymologia 33, 201-210. YARNELL,G. R., NELSONP. A. & WAGLES. R. (1966) Biochemical studies of the developing embryo-I. Metabolism of gluconeogenic precursors. Arch. B~chem. Biophw. 114, 539-542. YOUNGBURG G. E. & YOUNGBURG M. V.(1930) A system of blood phosphorus anaiysis. J. lab. Clin. 16, 158-166.