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Development of gluconeogenic enzymes in fetal sheep liver and kidney
DEVELOPMENTAL
BIOLOGY
52, 167-172 (1976)
Development
of Gluconeogenic Enzymes Sheep Liver and Kidney
ROGER E. STEVENSON,~ FRANK Program
in Pediatrics,
H. MORRISS, JR., EUGENE R. RODNEY HOWELL
W. ADCOCK, III, AND
The Uniuersity of Texas Health Science Center at Houston, Cullen Street, Houston, Texas 77025 Accepted
February
in Fetal
Medical
School, 6400 West
21,1976
In the sheep, the system of enzymes necessary for conversion of nonhexose substrates to glucose becomes active during late fetal life. Glucose-6phosphatase and fructose-1,6-diphosphatase, two of the four key gluconeogenic enzymes, appear in significant amounts between 100 and 120 days gestation. Phosphoenolpyruvate carboxykinase activity is comparable to mature animals as early as 45 days gestation. Two aminotransferases, necessary to allow amino acid access to the gluconeogenic pathway, likewise have substantial activity as early as 45 days gestation. Hence, the surge of glucose-6-phosphatase and fructose-1,6-diphosphatase at loo-120 days gestation makes possible the endogenous production of new glucose by fetal sheep at a time when the amount of glucose transferred from the maternal circulation is less than the total aerobic substrate utilized by the fetus. Both renal cortex and liver have similar developmental patterns for the gluconeogenic enzymes, although renal cortex generally shows greater activity than liver. This observation holds true for tissue from both fetal and mature animals.
urea excretion rate in fetal sheep to account for 25% of the fetal oxygen consumption through catabolism of amino acids (12). Comparisons of glucose and oxygen uptake by fetal brain and by fetal hindlimb preparations suggest that glucose alone can provide the total aerobic substrate in these tissues (16, 21). If these two tissue masses are typical of other fetal tissues, there would be a requirement for new glucose formation by the fetus. For amino acids to provide significant substrate for new glucose formation in the near-term fetus requires that fetal tissues have mature enzyme systems for transamination of amino acids and reversal of the glycolytic pathway (see Fig. 1). Four enzymes are essential for reversal of the glycolytic pathway, hence for gluconeogenesis from amino acids: phosphoenolpyruvate carboxykinase (PEP-CK), pyruvate carboxylase (PC), fructose-1,6-diphosphatase (FDP) , and glucose-6-phosphatase (G6P). Ballard and Oliver’s demonstration that
INTRODUCTION
Ruminants, in contrast to monogastric mammals, absorb little glucose from the gut and depend on nonhexose substrates as principal metabolic fuels (2, 3). In the mature animal, propionate and amino acids are the primary substrates for endogenous glucose production; glycerol, pyruvate, and lactate provide less significant sources of glucose production. In an analogous manner, the sheep fetus does not receive sufficient glucose from the maternal circulation to supply the total substrate required for aerobic metabolism during the final month of gestation (6, 14, 20). Amino acids have been proposed as the major source for the balance of aerobic substrate. This proposal is supported by the fact that propionate, the other major substrate, is promptly cleared from the maternal circulation as it passes through the liver (4, 9). Additionally, Gresham et al. have demonstrated a suf&iently high I Present Greenwood,
address: Greenwood Genetic South Carolina 29646.
Center, 167
Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
168
VOLUME 52, 1976
DEVELOPMENTAL BIOLOGY GLUCOSE
glucose-6-phosphntase (EC 3.1.3.9) GLUCOSE-&PHOSPHATE
FRUCTOSE-&PHOSPHATE ructose-t,F-diphosphatase (EC 3.1.3.11)
Pi L
FRUCTOSE-l,&DIPHOSPHATE glycerol
------+ t PHOSPHOENOL PYRUVATE
amino acids ------+ (aspartic acid) /-propionate~--amino acids (alanine)
phosphoenolpyruuate (EC 4.1.1.32)
: OXALOACETATE
carbozykinase
__a pymvate carboqlase (EC 6.4.1.1)
I PYRUVATE
-----___+ _/-
lactate
FIG. 1. Gluconeogenic gluconeogenic substrates
----
pathway (----+I.
indicating
key enzymes
liver slices from fetal sheep at 4 months gestation can convert [14Clpyruvate into glucose verifies that these key enzymes are active late in gestation (1). These authors also demonstrated significant FDP and G6P activities in sheep liver as early as 120-days gestation. Smith et al. (19) found mature sheep fetuses and l- to 7day-old lambs to have alanine and aspartate transaminases (GPT and GOT) present in the cytosolic fraction of both renal cortex and liver homogenates. In the present study, three key gluconeogenic enzymes and two aminotransferases have been assayed in liver and kidney specimens from sheep fetuses aged 44-145 days gestation and from lambs aged 1 and 4 days, Patterns of development of these enzymes during fetal life, relative enzyme activities in the two primary gluconeogenie tissues, correlation of enzyme activities with known fetal requirements for energy substrate, and comparison of fetal enzyme activities with those of mature sheep are presented.
(italics)
and points of entrance
MATERIALS
AND
of nonhexose
METHODS
Liver and kidney specimens were obtained from 17 fetuses of 44-145 days gestational age, from l- and 4-day-old nursing lambs, and from 2 nonpregnant and 10 pregnant ewes. All sheep were mixed Dorset and Western breeds. Maternal and fetal organ samples were obtained at cesarean section performed under barbiturate sedation or immediately following sacrifice of the ewe by decapitation. Samples were immediately frozen by Dry Ice and stored at -80°C until assayed. Gestational age was determined from fetal measurements of weight, crown-torump distance, and vertebral column length (8, 15). All fetal weights were appropriate for the assigned gestational ages. Fetuses designated “well-fed” were delivered from ewes whose dietary intakes of sweet feed2 were commensurate with normal fetal growth during pregnancy 2 Omolene, MO.
Ralston
Purina
Company,
St.
Louis,
STEVENSONET
AL.
Fetal Gluconeogenic
Enzymes
169
phosphatase equaled 30-40% of the total activity. Glucose-6-phosphatase. Hers method for G6P measures inorganic phosphate released from glucose-6-phosphate after incubation with homogenate at pH 6.5 and 37°C (13). The contribution of nonspecific phosphatases was eliminated by running a blank in which the homogenate had been incubated at pH 5.0 for 5 min. The reaction contained glucose-6-phosphate 50 n&f, cacodylate buffer (pH 6.5) 75 m&f, and homogenate in a total volume of 0.2 ml. The reaction was terminated at 50 min with 0.5 ml of trichloracetic acid (0.25 M), and inorganic phosphate was determined by the Phosphoenolpyruvate carboxykinase. PEP-CK was assayed by following the rate method of Fiske and SubbaRow. Nonspeof NADH oxidation in the following reac- cific phosphatase was subtracted from the tion mixture: Tris-Cl buffer (pH 7.5) 100 total to determine specific glucose-6-phosm&f, KHCO, 50 n-&f, MnCl, 1.0 mM, GSH phatase. Glutamate-oxaloacetate aminotrans2.0 n-&f, GDP 1.25 mM, NADH 0.15 n-&f, PEP 1.25 mM, and MDH 5 units. Blanks ferase. GOT activity was determined by measuring oxaloacetate formed from aswithout KHCO, were run simultaneously. The total volume was 1.O ml and the parate and a-oxoglutarate, utilizing malic NADH oxidation was read at 340 nm at dehydrogenase and NADH as an enzyme 30°C for 10 min. Under these conditions, indicator reaction (5). The reaction mixeach micromole of NADH oxidized indi- ture contained NADH 0.4 n-&f, asparate cates carboxylation of 1 pmol of PEP to 218 n-&f, phosphate buffer (pH 7.6) 87 n-&f, MDH 5 units, and a-oxoglutarate 6.7 mM, form oxalacetate (7). Fructose-l ,6-diphosphatase. FDP activ- in a total volume of 1.5 ml. The assay ity was determined by quantitating the temperature was 25°C. The reaction was inorganic phosphate released from fruc- initiated with a-oxoglutarate after equitose-1,6-diphosphate substrate by incuba- librium for 5 to 10 min. The oxidation of tion with homogenate at pH 9.4 (17). The NADH was determined by following abreaction mixture, containing fructose-1,6- sorption at 340 nm for 10 min. Under these diphosphate 5 n-&f, glycine buffer (pH 9.4) conditions 1 pmol of NADH oxidation indi100 mM, and MgCl, 5.0 mM in 0.2 ml of cates 1 pmol of asparate converted to oxaltotal volume, was incubated at 37°C for 60 oacetate. min. The reaction was terminated by addiGlutamate-pyruvate aminotransferase. tion of 0.5 ml of trichloracetic acid (0.25 The methodology for GPT is essentially the M). Phosphate was determined by the same as for GOT (5). The reaction mixture method of Fiske and SubbaRow (11) and contained L-alanine 98 mM, phosphate compared to a phosphate standard run in buffer (pH 7.6) 89 mM, a-oxoglutarate 6.7 the same manner. Elimination of error n-&f, NADH 0.16 mM, and LDH 5 units, in a total volume of 1.5 ml. In this LDHfrom fructose-1,8diphosphate hydrolysis by other phosphatases was not possible. coupled reaction, each micromole of By substituting fructose-6-phosphate as NADH oxidized indicates 1 Fmol of alathe substrate, the amount of nonspecific nine converted to pyruvate.
(23). Fetuses designated “fasting” were delivered from ewes that had been fasted for lV2 days prior to sacrifice or whose nutrition was less than optimal for several days prior to sacrifice. Enzyme assays were performed on 1% tissue homogenates prepared in cold distilled water using a Ten Broeck hand homogenizer. Cytosolic and mitochondrial fractions were not assayed separately in this study. Protein concentrations in the homogenate were determined by the fluram method (22), and enzyme activities were assayed according to the following methods.
170
DEVELOPMENTAL BIOLOGY
Linear regression analyses by the least squares method were performed for the activity of each enzyme against gestational age. The significance of the correlation coefficient for each regression was determined from tables of percentiles of the distribution of r whenP = 0. Differences in group mean enzyme activities were measured by the unpaired Student’s t test (10). RESULTS
Mean activities for the gluconeogenic enzymes and two aminotransferases in fetal and adult liver and kidney are presented in Table 1. Individual values for TABLE
1
ACTIVITIES OF KEY GLUCONE~GENIC ENZYMES AND Two AMINOTRANSFERASES IN FETAL AND ADULT SHEEP LIVER AND KIDNEY Tissue
Enzyme
Liver
G6P FDP PEP-CK GOT GPT G6P FDP PEP-CK GOT GPT
Kidney
* Enzyme activity
Fetal Adult (5 r SE. wM/min/g protein)
* 10.8 249.0 61.1
59.4 152.5 55.0
* k + & * * k k k
1.1 16.3 11.1
8.0 11.2 6.5
65.3 46.7 6.9 336.9 117.9 70.8 93.1 46.2 166.8 122.8
+ f f ? + + f + + +
increases with gestational
4.6 5.2 1.4 65.2 11.0 13.4 5.2 7.4 19.9 10.9 age.
FIG. 2. Glucose-6-phosphatase activity in (a) liver ages and from l- and I-day-old lambs. The stippled mature pregnant and nonpregnant ewes. Key: Circle symbol = fetus of well-fed ewe; open symbol = fetus
VOLUME 52, 1976
fetal G6P and FDP, the only enzymes that changed significantly during gestation, are plotted in Figs. 2 and 3. For comparison the mean + SE enzyme activities for pregnant and nonpregnant adult ewes are shown as overlays on the same graphs. There was no apparent difference in enzyme activities between well-fed and fasting animals of comparable gestational age. Glucose-6-phosphatase. G6P activity increased from very low levels during the first half of gestation to levels comparable to or higher than adult levels following delivery (Figs. 2a, b). The surge of enzyme activity occurred between 100 and 120 days gestation, and was similar in magnitude and timing in both liver and kidney. Fructose-l ,gdiphosphatase. A similar surge of total FDP activity was observed in fetal kidney specimens (Fig. 3b). Fetal liver FDP activity increased throughout pregnancy (Fig. 3a). Maternal kidney specimens showed greater FDP activity than did liver samples (P < 0.005). Fetal and neonatal kidney FDP activity did not reach maternal levels, whereas fetal liver samples had FDP activity comparable to adult values by 100 days gestation. Phosphoenolpyruvate
carboxykinase.
Fetal liver PEP-CK activity was significantly greater than adult liver PEP-CK
and (b) kidney from fetal sheep of varying gestational overlay indicates the mean ? SE for G6P activity in = singleton fetus or lamb; triangle = twin fetus; solid of fasting ewe.
STEVENSON ET AL.
Fetal Gluconeogenic
171
Enzymes
0
.:,
a,. ‘A A
.a
b
A
A
l
FIG. 3. Total fructose-1,6-diphosphatase activity in (a) liver and (b) kidney from fetal sheep of varying gestational ages and from l- and 4-day-old lambs. The stippled overlay indicates the mean + SE for FDP activity in mature pregnant and nonpregnant ewes. Nonspecific phosphatase (30-40% of total) has not been subtracted from the total FDP activity. Key: Circle = singleton fetus or lamb; triangle = twin fetus; solid symbol = fetus of well-fed ewe; open symbol = fetus of fasting ewe.
activity (P < 0.05). Although fetal kidney PEP-CK activity tended to be greater than adult kidney PEP-CK activity in midgestation, the mean values were not significantly different. For both fetal and adult animals PEPCK activity was greater in kidney than in liver (P < 0.005). No significant alterations in levels occurred with advancing gestational age. Aspartate aminotransferase. Adults and fetuses had similar GOT levels in kidney specimens. GOT levels in liver were somewhat higher for both. No significant change with gestation was observed. Alanine aminotransferase. In liver and in kidney GPT values for fetal specimens were lower than for adult (P < 0.005). Fetal enzyme activity was comparable in the two tissues. Fetal GPT activity decreased in kidney throughout pregnancy, an observation of low statistical significance (P < 0.05). DISCUSSION
Utilization of the gluconeogenic pathway permits fetal conversion of nonhexose substances, principally amino acids, propionate, pyruvate, lactate, and glycerol to glucose (Fig. 1). Additionally, the complete degradation of glycogen, known to be stored in fetal liver in significant quantities during the final month of pregnancy,
to free glucose depends upon G6P activity (18). Fetal liver and kidney have limited in vitro ability for endogenous glucose production prior to 100 days gestational age, primarily due to low levels of G6P and FDP activities. As gestation approaches term, the activities of these key gluconeogenie enzymes in fetal sheep liver and kidney achieve levels comparable to those in the adult ewe. Both liver and kidney, the major organs with gluconeogenic activity, appear similar in respect to development of these two enzyme systems. Other potentially gluconeogenic fetal tissues that may add to the total new glucose production have received little attention. The contribution of fetal gut mucosa is not known for various gestational ages. The gluconeogenic pathway in placenta is interrupted by the near total absence of G6P activity throughout pregnancy.3 In the fetus, the activities of the gluconeogenic enzymes and GPT in renal cortex equal or exceed the activities in liver (Table 1). GOT alone is less in the kidney. These values parallel those in adult ewe liver and kidney quite closely. In mature sheep, propionate provides approximately 70% of the substrate for new glucose formation. Amino acids and propionate together provide approximately 90%. With efficient removal of propionate from the 3 Unpublished observations by the authors.
172
DEVELOPMENTAL BIOLOC Y
portal circulation by mature sheep, one might anticipate that amino acids from maternal serum comprise a more important source of gluconeogenic substrate for the fetus. Urea production by the fetus is consistent with utilization of amino acids for this purpose. This study does not confirm that amino acids are the substrate utilized in late pregnancy, but rather that the capacity to convert amino acids to glucose is present. By the same measure, any other substrate that can find its way to the gluconeogenic pathway (propionate, pyruvate, lactate, glycerol, other hexoses) may be utilized as well. REFERENCES 1. BALLARD, F. J., and OLIVER, I. T. (1965). Carbohydrate metabolism in liver from fetal and neonatal sheep. Biochem. J. 95, 191-200. 2. BALLARD, F. J., HANSON, R. W., and KRONFELD, D. S. (1969). Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed. Proc. 28, 218-231. 3. BERGMAN, E. N. (1973). Glucose metabolism in ruminants as related to hypoglycemia and ketosis. The Cornell Veterinarian 63, 341-382. 4. BERGMAN, E. N., and WOLFF, J. E. (1971). Metabolism of volatile fatty acids by liver and portal-drained viscera in sheep. Amer. J. Physiol. 221, 586-592. 5. BERGMEYER, H.-U., and BERNT, E. (1965). Glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase. In “Methods of Enzymatic Analysis” (H.-U. Bergmeyer, ed.1, pp. 837-843. Academic Press, New York. 6. BOYD, R. D. H., MORRISS, JR., F. H., MESCHIA, G., MAKOWSKI, E. L., and BATTAGLIA, F. C. (1973). Growth of glucose and oxygen uptakes by fetuses of fed and starved ewes. Amer. J. Physiol. 225, 897-902. 7. CHANG, H.-C., and LANE, M. D. (1966). The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J. Biol. Chem. 241, 2413-2420. 8. CLOETE, J. H. L. (1939). Prenatal growth in the Merino sheep. Onderstepoort J. of Veterinary Science and Animal Industry 13, 417-557. 9. COOK, R. M., and MILLER, L. D. (1965). Utilization of volatile fatty acids in ruminants. I. Removal from portal blood by the liver. J. Dairy Science 48, 1339-1345. 10. DIXON, W. J., and MASSEY, F. J. (19691. Zn “Introduction to Statistical Analysis.” 3rd ed.,
VOLUME 52, 1976 pp. 114, 196, 569. McGraw-Hill, New York. 11. FISKE, C. H., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. 12. GRESHAM, E. L., JAMES, E. J., RAYE, J. R., BATTAGLIA, F. C., MAKOWSKI, E. J., and MESCHIA, G. (1972). Production and excretion of urea by the fetal lamb. Pediatrics 50, 372-379. 13. HERS, H. G. (1964). Glycogen storage disease. In “Advances in Metabolic Disorders” (R. Levine, and R. Luft, eds.), p. l-44. Academic Press, New York. 14. JAMES, E., RAYE, J. R., GRESHAM, E. L., MAKOWSKI, E. L., MESCHIA, G., and BATTAGLIA, F. C. (1972). Fetal oxygen consumption, carbon dioxide production, and glucose uptake in a chronic sheep preparation. Pediatrics 50, 361-371. 15. KULHANEK, J. F. (1972). Gestational changes in DNA content and urea permeability of the sheep placenta and their relationship to fetal growth. Ph. D. Thesis, University of Colorado. 16. MORRISS, JR., F. H., BOYD, R. D. H., MAKOWSKI, E. L., MESCHIA, G., and BATTAGLIA, F. C. (1973). Glucose/oxygen quotients across the hindlimb of fetal lambs. Pediat. Res. 7, 794797. 17. PONTREMOLI, S. (1966). Fructose-1,6-diphosphatase. In “Methods in Enzymology” (S. P. Colowick, and N. 0. Kaplan, eds.), Vol. XI, pp. 625-631. Academic Press, New York. 18. SHELLEY, H. J. (1961). Glycogen reserves and their changes at birth. Brit. Med. Bull. 17, 137-143. 19. SMITH, E. M., SHEPHERD, D. A. L., and JEACOCK, M. K. (1969). Activity of aminotransferases in the tissues of developing lambs. Biothem. J. 115, 39P-40P. 20. TSOULOS, N. G., COLWILL, J. R., BATTAGLIA, F. C., MAKOWSKI, E. L., and MESCHIA, G. (1971). Comparison of glucose, fructose, and 0, uptakes by fetuses of fed and starved ewes. Amer. J. Physiol. 221, 234-237. 21. TSOULOS, N. G., SCHNEIDER, J. M., COLWILL, J. R., MESCHIA, G., MAKOWSKI, E. L., and BATTAGLIA, F. C. (1972). Cerebral glucose utilization during aerobic metabolism in fetal sheep. Pediat. Res. 6, 182-186. 22. UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIRMAN, W., LEIMGRUBER, W., and WEIGELE, M. (1972). Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 177, 871-872. 23. WALLACE, L. R. (1948). The growth of lambs before and after birth in relation to the level of nutrition. Part III. J. Agric. Sci. 38, 367401; Fig. 61, p. 368.
BIOLOGY
52, 167-172 (1976)
Development
of Gluconeogenic Enzymes Sheep Liver and Kidney
ROGER E. STEVENSON,~ FRANK Program
in Pediatrics,
H. MORRISS, JR., EUGENE R. RODNEY HOWELL
W. ADCOCK, III, AND
The Uniuersity of Texas Health Science Center at Houston, Cullen Street, Houston, Texas 77025 Accepted
February
in Fetal
Medical
School, 6400 West
21,1976
In the sheep, the system of enzymes necessary for conversion of nonhexose substrates to glucose becomes active during late fetal life. Glucose-6phosphatase and fructose-1,6-diphosphatase, two of the four key gluconeogenic enzymes, appear in significant amounts between 100 and 120 days gestation. Phosphoenolpyruvate carboxykinase activity is comparable to mature animals as early as 45 days gestation. Two aminotransferases, necessary to allow amino acid access to the gluconeogenic pathway, likewise have substantial activity as early as 45 days gestation. Hence, the surge of glucose-6-phosphatase and fructose-1,6-diphosphatase at loo-120 days gestation makes possible the endogenous production of new glucose by fetal sheep at a time when the amount of glucose transferred from the maternal circulation is less than the total aerobic substrate utilized by the fetus. Both renal cortex and liver have similar developmental patterns for the gluconeogenic enzymes, although renal cortex generally shows greater activity than liver. This observation holds true for tissue from both fetal and mature animals.
urea excretion rate in fetal sheep to account for 25% of the fetal oxygen consumption through catabolism of amino acids (12). Comparisons of glucose and oxygen uptake by fetal brain and by fetal hindlimb preparations suggest that glucose alone can provide the total aerobic substrate in these tissues (16, 21). If these two tissue masses are typical of other fetal tissues, there would be a requirement for new glucose formation by the fetus. For amino acids to provide significant substrate for new glucose formation in the near-term fetus requires that fetal tissues have mature enzyme systems for transamination of amino acids and reversal of the glycolytic pathway (see Fig. 1). Four enzymes are essential for reversal of the glycolytic pathway, hence for gluconeogenesis from amino acids: phosphoenolpyruvate carboxykinase (PEP-CK), pyruvate carboxylase (PC), fructose-1,6-diphosphatase (FDP) , and glucose-6-phosphatase (G6P). Ballard and Oliver’s demonstration that
INTRODUCTION
Ruminants, in contrast to monogastric mammals, absorb little glucose from the gut and depend on nonhexose substrates as principal metabolic fuels (2, 3). In the mature animal, propionate and amino acids are the primary substrates for endogenous glucose production; glycerol, pyruvate, and lactate provide less significant sources of glucose production. In an analogous manner, the sheep fetus does not receive sufficient glucose from the maternal circulation to supply the total substrate required for aerobic metabolism during the final month of gestation (6, 14, 20). Amino acids have been proposed as the major source for the balance of aerobic substrate. This proposal is supported by the fact that propionate, the other major substrate, is promptly cleared from the maternal circulation as it passes through the liver (4, 9). Additionally, Gresham et al. have demonstrated a suf&iently high I Present Greenwood,
address: Greenwood Genetic South Carolina 29646.
Center, 167
Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved.
168
VOLUME 52, 1976
DEVELOPMENTAL BIOLOGY GLUCOSE
glucose-6-phosphntase (EC 3.1.3.9) GLUCOSE-&PHOSPHATE
FRUCTOSE-&PHOSPHATE ructose-t,F-diphosphatase (EC 3.1.3.11)
Pi L
FRUCTOSE-l,&DIPHOSPHATE glycerol
------+ t PHOSPHOENOL PYRUVATE
amino acids ------+ (aspartic acid) /-propionate~--amino acids (alanine)
phosphoenolpyruuate (EC 4.1.1.32)
: OXALOACETATE
carbozykinase
__a pymvate carboqlase (EC 6.4.1.1)
I PYRUVATE
-----___+ _/-
lactate
FIG. 1. Gluconeogenic gluconeogenic substrates
----
pathway (----+I.
indicating
key enzymes
liver slices from fetal sheep at 4 months gestation can convert [14Clpyruvate into glucose verifies that these key enzymes are active late in gestation (1). These authors also demonstrated significant FDP and G6P activities in sheep liver as early as 120-days gestation. Smith et al. (19) found mature sheep fetuses and l- to 7day-old lambs to have alanine and aspartate transaminases (GPT and GOT) present in the cytosolic fraction of both renal cortex and liver homogenates. In the present study, three key gluconeogenic enzymes and two aminotransferases have been assayed in liver and kidney specimens from sheep fetuses aged 44-145 days gestation and from lambs aged 1 and 4 days, Patterns of development of these enzymes during fetal life, relative enzyme activities in the two primary gluconeogenie tissues, correlation of enzyme activities with known fetal requirements for energy substrate, and comparison of fetal enzyme activities with those of mature sheep are presented.
(italics)
and points of entrance
MATERIALS
AND
of nonhexose
METHODS
Liver and kidney specimens were obtained from 17 fetuses of 44-145 days gestational age, from l- and 4-day-old nursing lambs, and from 2 nonpregnant and 10 pregnant ewes. All sheep were mixed Dorset and Western breeds. Maternal and fetal organ samples were obtained at cesarean section performed under barbiturate sedation or immediately following sacrifice of the ewe by decapitation. Samples were immediately frozen by Dry Ice and stored at -80°C until assayed. Gestational age was determined from fetal measurements of weight, crown-torump distance, and vertebral column length (8, 15). All fetal weights were appropriate for the assigned gestational ages. Fetuses designated “well-fed” were delivered from ewes whose dietary intakes of sweet feed2 were commensurate with normal fetal growth during pregnancy 2 Omolene, MO.
Ralston
Purina
Company,
St.
Louis,
STEVENSONET
AL.
Fetal Gluconeogenic
Enzymes
169
phosphatase equaled 30-40% of the total activity. Glucose-6-phosphatase. Hers method for G6P measures inorganic phosphate released from glucose-6-phosphate after incubation with homogenate at pH 6.5 and 37°C (13). The contribution of nonspecific phosphatases was eliminated by running a blank in which the homogenate had been incubated at pH 5.0 for 5 min. The reaction contained glucose-6-phosphate 50 n&f, cacodylate buffer (pH 6.5) 75 m&f, and homogenate in a total volume of 0.2 ml. The reaction was terminated at 50 min with 0.5 ml of trichloracetic acid (0.25 M), and inorganic phosphate was determined by the Phosphoenolpyruvate carboxykinase. PEP-CK was assayed by following the rate method of Fiske and SubbaRow. Nonspeof NADH oxidation in the following reac- cific phosphatase was subtracted from the tion mixture: Tris-Cl buffer (pH 7.5) 100 total to determine specific glucose-6-phosm&f, KHCO, 50 n-&f, MnCl, 1.0 mM, GSH phatase. Glutamate-oxaloacetate aminotrans2.0 n-&f, GDP 1.25 mM, NADH 0.15 n-&f, PEP 1.25 mM, and MDH 5 units. Blanks ferase. GOT activity was determined by measuring oxaloacetate formed from aswithout KHCO, were run simultaneously. The total volume was 1.O ml and the parate and a-oxoglutarate, utilizing malic NADH oxidation was read at 340 nm at dehydrogenase and NADH as an enzyme 30°C for 10 min. Under these conditions, indicator reaction (5). The reaction mixeach micromole of NADH oxidized indi- ture contained NADH 0.4 n-&f, asparate cates carboxylation of 1 pmol of PEP to 218 n-&f, phosphate buffer (pH 7.6) 87 n-&f, MDH 5 units, and a-oxoglutarate 6.7 mM, form oxalacetate (7). Fructose-l ,6-diphosphatase. FDP activ- in a total volume of 1.5 ml. The assay ity was determined by quantitating the temperature was 25°C. The reaction was inorganic phosphate released from fruc- initiated with a-oxoglutarate after equitose-1,6-diphosphate substrate by incuba- librium for 5 to 10 min. The oxidation of tion with homogenate at pH 9.4 (17). The NADH was determined by following abreaction mixture, containing fructose-1,6- sorption at 340 nm for 10 min. Under these diphosphate 5 n-&f, glycine buffer (pH 9.4) conditions 1 pmol of NADH oxidation indi100 mM, and MgCl, 5.0 mM in 0.2 ml of cates 1 pmol of asparate converted to oxaltotal volume, was incubated at 37°C for 60 oacetate. min. The reaction was terminated by addiGlutamate-pyruvate aminotransferase. tion of 0.5 ml of trichloracetic acid (0.25 The methodology for GPT is essentially the M). Phosphate was determined by the same as for GOT (5). The reaction mixture method of Fiske and SubbaRow (11) and contained L-alanine 98 mM, phosphate compared to a phosphate standard run in buffer (pH 7.6) 89 mM, a-oxoglutarate 6.7 the same manner. Elimination of error n-&f, NADH 0.16 mM, and LDH 5 units, in a total volume of 1.5 ml. In this LDHfrom fructose-1,8diphosphate hydrolysis by other phosphatases was not possible. coupled reaction, each micromole of By substituting fructose-6-phosphate as NADH oxidized indicates 1 Fmol of alathe substrate, the amount of nonspecific nine converted to pyruvate.
(23). Fetuses designated “fasting” were delivered from ewes that had been fasted for lV2 days prior to sacrifice or whose nutrition was less than optimal for several days prior to sacrifice. Enzyme assays were performed on 1% tissue homogenates prepared in cold distilled water using a Ten Broeck hand homogenizer. Cytosolic and mitochondrial fractions were not assayed separately in this study. Protein concentrations in the homogenate were determined by the fluram method (22), and enzyme activities were assayed according to the following methods.
170
DEVELOPMENTAL BIOLOGY
Linear regression analyses by the least squares method were performed for the activity of each enzyme against gestational age. The significance of the correlation coefficient for each regression was determined from tables of percentiles of the distribution of r whenP = 0. Differences in group mean enzyme activities were measured by the unpaired Student’s t test (10). RESULTS
Mean activities for the gluconeogenic enzymes and two aminotransferases in fetal and adult liver and kidney are presented in Table 1. Individual values for TABLE
1
ACTIVITIES OF KEY GLUCONE~GENIC ENZYMES AND Two AMINOTRANSFERASES IN FETAL AND ADULT SHEEP LIVER AND KIDNEY Tissue
Enzyme
Liver
G6P FDP PEP-CK GOT GPT G6P FDP PEP-CK GOT GPT
Kidney
* Enzyme activity
Fetal Adult (5 r SE. wM/min/g protein)
* 10.8 249.0 61.1
59.4 152.5 55.0
* k + & * * k k k
1.1 16.3 11.1
8.0 11.2 6.5
65.3 46.7 6.9 336.9 117.9 70.8 93.1 46.2 166.8 122.8
+ f f ? + + f + + +
increases with gestational
4.6 5.2 1.4 65.2 11.0 13.4 5.2 7.4 19.9 10.9 age.
FIG. 2. Glucose-6-phosphatase activity in (a) liver ages and from l- and I-day-old lambs. The stippled mature pregnant and nonpregnant ewes. Key: Circle symbol = fetus of well-fed ewe; open symbol = fetus
VOLUME 52, 1976
fetal G6P and FDP, the only enzymes that changed significantly during gestation, are plotted in Figs. 2 and 3. For comparison the mean + SE enzyme activities for pregnant and nonpregnant adult ewes are shown as overlays on the same graphs. There was no apparent difference in enzyme activities between well-fed and fasting animals of comparable gestational age. Glucose-6-phosphatase. G6P activity increased from very low levels during the first half of gestation to levels comparable to or higher than adult levels following delivery (Figs. 2a, b). The surge of enzyme activity occurred between 100 and 120 days gestation, and was similar in magnitude and timing in both liver and kidney. Fructose-l ,gdiphosphatase. A similar surge of total FDP activity was observed in fetal kidney specimens (Fig. 3b). Fetal liver FDP activity increased throughout pregnancy (Fig. 3a). Maternal kidney specimens showed greater FDP activity than did liver samples (P < 0.005). Fetal and neonatal kidney FDP activity did not reach maternal levels, whereas fetal liver samples had FDP activity comparable to adult values by 100 days gestation. Phosphoenolpyruvate
carboxykinase.
Fetal liver PEP-CK activity was significantly greater than adult liver PEP-CK
and (b) kidney from fetal sheep of varying gestational overlay indicates the mean ? SE for G6P activity in = singleton fetus or lamb; triangle = twin fetus; solid of fasting ewe.
STEVENSON ET AL.
Fetal Gluconeogenic
171
Enzymes
0
.:,
a,. ‘A A
.a
b
A
A
l
FIG. 3. Total fructose-1,6-diphosphatase activity in (a) liver and (b) kidney from fetal sheep of varying gestational ages and from l- and 4-day-old lambs. The stippled overlay indicates the mean + SE for FDP activity in mature pregnant and nonpregnant ewes. Nonspecific phosphatase (30-40% of total) has not been subtracted from the total FDP activity. Key: Circle = singleton fetus or lamb; triangle = twin fetus; solid symbol = fetus of well-fed ewe; open symbol = fetus of fasting ewe.
activity (P < 0.05). Although fetal kidney PEP-CK activity tended to be greater than adult kidney PEP-CK activity in midgestation, the mean values were not significantly different. For both fetal and adult animals PEPCK activity was greater in kidney than in liver (P < 0.005). No significant alterations in levels occurred with advancing gestational age. Aspartate aminotransferase. Adults and fetuses had similar GOT levels in kidney specimens. GOT levels in liver were somewhat higher for both. No significant change with gestation was observed. Alanine aminotransferase. In liver and in kidney GPT values for fetal specimens were lower than for adult (P < 0.005). Fetal enzyme activity was comparable in the two tissues. Fetal GPT activity decreased in kidney throughout pregnancy, an observation of low statistical significance (P < 0.05). DISCUSSION
Utilization of the gluconeogenic pathway permits fetal conversion of nonhexose substances, principally amino acids, propionate, pyruvate, lactate, and glycerol to glucose (Fig. 1). Additionally, the complete degradation of glycogen, known to be stored in fetal liver in significant quantities during the final month of pregnancy,
to free glucose depends upon G6P activity (18). Fetal liver and kidney have limited in vitro ability for endogenous glucose production prior to 100 days gestational age, primarily due to low levels of G6P and FDP activities. As gestation approaches term, the activities of these key gluconeogenie enzymes in fetal sheep liver and kidney achieve levels comparable to those in the adult ewe. Both liver and kidney, the major organs with gluconeogenic activity, appear similar in respect to development of these two enzyme systems. Other potentially gluconeogenic fetal tissues that may add to the total new glucose production have received little attention. The contribution of fetal gut mucosa is not known for various gestational ages. The gluconeogenic pathway in placenta is interrupted by the near total absence of G6P activity throughout pregnancy.3 In the fetus, the activities of the gluconeogenic enzymes and GPT in renal cortex equal or exceed the activities in liver (Table 1). GOT alone is less in the kidney. These values parallel those in adult ewe liver and kidney quite closely. In mature sheep, propionate provides approximately 70% of the substrate for new glucose formation. Amino acids and propionate together provide approximately 90%. With efficient removal of propionate from the 3 Unpublished observations by the authors.
172
DEVELOPMENTAL BIOLOC Y
portal circulation by mature sheep, one might anticipate that amino acids from maternal serum comprise a more important source of gluconeogenic substrate for the fetus. Urea production by the fetus is consistent with utilization of amino acids for this purpose. This study does not confirm that amino acids are the substrate utilized in late pregnancy, but rather that the capacity to convert amino acids to glucose is present. By the same measure, any other substrate that can find its way to the gluconeogenic pathway (propionate, pyruvate, lactate, glycerol, other hexoses) may be utilized as well. REFERENCES 1. BALLARD, F. J., and OLIVER, I. T. (1965). Carbohydrate metabolism in liver from fetal and neonatal sheep. Biochem. J. 95, 191-200. 2. BALLARD, F. J., HANSON, R. W., and KRONFELD, D. S. (1969). Gluconeogenesis and lipogenesis in tissue from ruminant and nonruminant animals. Fed. Proc. 28, 218-231. 3. BERGMAN, E. N. (1973). Glucose metabolism in ruminants as related to hypoglycemia and ketosis. The Cornell Veterinarian 63, 341-382. 4. BERGMAN, E. N., and WOLFF, J. E. (1971). Metabolism of volatile fatty acids by liver and portal-drained viscera in sheep. Amer. J. Physiol. 221, 586-592. 5. BERGMEYER, H.-U., and BERNT, E. (1965). Glutamate-oxaloacetate transaminase and glutamate-pyruvate transaminase. In “Methods of Enzymatic Analysis” (H.-U. Bergmeyer, ed.1, pp. 837-843. Academic Press, New York. 6. BOYD, R. D. H., MORRISS, JR., F. H., MESCHIA, G., MAKOWSKI, E. L., and BATTAGLIA, F. C. (1973). Growth of glucose and oxygen uptakes by fetuses of fed and starved ewes. Amer. J. Physiol. 225, 897-902. 7. CHANG, H.-C., and LANE, M. D. (1966). The enzymatic carboxylation of phosphoenolpyruvate. II. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase. J. Biol. Chem. 241, 2413-2420. 8. CLOETE, J. H. L. (1939). Prenatal growth in the Merino sheep. Onderstepoort J. of Veterinary Science and Animal Industry 13, 417-557. 9. COOK, R. M., and MILLER, L. D. (1965). Utilization of volatile fatty acids in ruminants. I. Removal from portal blood by the liver. J. Dairy Science 48, 1339-1345. 10. DIXON, W. J., and MASSEY, F. J. (19691. Zn “Introduction to Statistical Analysis.” 3rd ed.,
VOLUME 52, 1976 pp. 114, 196, 569. McGraw-Hill, New York. 11. FISKE, C. H., and SUBBAROW, Y. (1925). The calorimetric determination of phosphorus. J. Biol. Chem. 66, 375-400. 12. GRESHAM, E. L., JAMES, E. J., RAYE, J. R., BATTAGLIA, F. C., MAKOWSKI, E. J., and MESCHIA, G. (1972). Production and excretion of urea by the fetal lamb. Pediatrics 50, 372-379. 13. HERS, H. G. (1964). Glycogen storage disease. In “Advances in Metabolic Disorders” (R. Levine, and R. Luft, eds.), p. l-44. Academic Press, New York. 14. JAMES, E., RAYE, J. R., GRESHAM, E. L., MAKOWSKI, E. L., MESCHIA, G., and BATTAGLIA, F. C. (1972). Fetal oxygen consumption, carbon dioxide production, and glucose uptake in a chronic sheep preparation. Pediatrics 50, 361-371. 15. KULHANEK, J. F. (1972). Gestational changes in DNA content and urea permeability of the sheep placenta and their relationship to fetal growth. Ph. D. Thesis, University of Colorado. 16. MORRISS, JR., F. H., BOYD, R. D. H., MAKOWSKI, E. L., MESCHIA, G., and BATTAGLIA, F. C. (1973). Glucose/oxygen quotients across the hindlimb of fetal lambs. Pediat. Res. 7, 794797. 17. PONTREMOLI, S. (1966). Fructose-1,6-diphosphatase. In “Methods in Enzymology” (S. P. Colowick, and N. 0. Kaplan, eds.), Vol. XI, pp. 625-631. Academic Press, New York. 18. SHELLEY, H. J. (1961). Glycogen reserves and their changes at birth. Brit. Med. Bull. 17, 137-143. 19. SMITH, E. M., SHEPHERD, D. A. L., and JEACOCK, M. K. (1969). Activity of aminotransferases in the tissues of developing lambs. Biothem. J. 115, 39P-40P. 20. TSOULOS, N. G., COLWILL, J. R., BATTAGLIA, F. C., MAKOWSKI, E. L., and MESCHIA, G. (1971). Comparison of glucose, fructose, and 0, uptakes by fetuses of fed and starved ewes. Amer. J. Physiol. 221, 234-237. 21. TSOULOS, N. G., SCHNEIDER, J. M., COLWILL, J. R., MESCHIA, G., MAKOWSKI, E. L., and BATTAGLIA, F. C. (1972). Cerebral glucose utilization during aerobic metabolism in fetal sheep. Pediat. Res. 6, 182-186. 22. UDENFRIEND, S., STEIN, S., BOHLEN, P., DAIRMAN, W., LEIMGRUBER, W., and WEIGELE, M. (1972). Fluorescamine: A reagent for assay of amino acids, peptides, proteins, and primary amines in the picomole range. Science 177, 871-872. 23. WALLACE, L. R. (1948). The growth of lambs before and after birth in relation to the level of nutrition. Part III. J. Agric. Sci. 38, 367401; Fig. 61, p. 368.