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Enzymes of carbohydrate and lipid metabolism in the placenta and liver of pregnant rats
424
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26952
ENZYMES OF CARBOHYDRATE AND L I P I D METABOLISM IN T H E PLACENTA AND LIVER OF PREGNANT RATS
Y O R A M Z. D I A M A N T AND E L E A Z A R S H A F R I R
Departments of Obstetrics and Gynecology and Clinical Biochemistry, Hadassa University Hospital and Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Received J u l y 2oth, 1972)
SUMMARY
Changes in the activity of enzymes involved in glycolysis, gluconeogenesis and lipogenesis during the last days of gestation were determined in the placenta and liver of pregnant rats and expressed in relation to the cytoplasmic protein content of these tissues. On Day 15 of gestation the activity of pyruvate kinase (EC 2.7.1.4o) in the placenta was considerably higher than in the liver, whereas the activity of placental enzymes associated with gluconeogenesis was much lower than in the liver. This indicated that the placental capacity of glucose utilization is preponderant over gluconeogenesis. The activity of enzymes associated with lipid synthesis was lower in the placenta in comparison to the liver. However, the activity of enzymes of NADPH generation and of ATP citrate lyase (EC 4.1.3.8) in relation to that of acetyl-CoA carboxylase (EC 6.4.1.2 ) was markedly higher in the placenta than in the liver. This was suggestive of the importance of synthesis of lipids other than fatty acids in the placenta. With the progress of gestation a decrease in the activity of most placental enzymes was observed, particularly in that of pyruvate kinase. In the liver the activity of enzymes of NADPH generation and of ATP citrate lyase also decreased. The possible background of these changes in enzyme activity in the liver and placenta is discussed.
INTRODUCTION
The metabolism of carbohydrates and lipids in the placenta provides the requirements of energy for the synthesis of a range of sterols and steroid hormones, the active transport of metabolites and other functions which are assumed to be auxilliary to the fetal liver. Enzymatic pathways are known to be present for the conversion of glucose to glycogen or its degradation in the glycolytic pathway and in the hexose monophosphate shunt 1-5. Significant production of glucose from pyruvate was observed in human placenta in the early stage of gestation 5. The decline in placental glucose-6-phosBiochim. Biophys. Acta, 279 (1972) 424-43 o
E N Z Y M E S IN P L A C E N T A A N D L I V E R O F P R E G N A N T R A T S
425
phatase activity with gestationS, e was considered to explain the absence of significant gluconeogenesis at term. On the other hand, the small rate of glucose and fructose production from labeled pyruvate was found not to differ in the young or term human placentasL It was attributed to nonspecific hexose phosphate hydrolysis by the abundant placental alkaline phosphatase~,L The quantity of lipid in the placenta is small s. Placental triglycerides and phospholipids are formed from esterification of fatty acids either taken up from maternal blood or synthesized de novo% 1°. Basic enzymatic steps of this pathway have been investigated in the placenta by Hosoya et al. ~. With regard to hepatic carbohydrate and fat metabolism in pregnancy, an enhancement in glycolysis with a concomitant reduction in oxidative metabolism was suggested 12. No changes in liver gluconeogenesis or glycogen synthesis have been observed in pregnant fed animals as compared to normal controls t3. An increased incorporation of I2-14CJpyruvate ~ and EI-14C]acetate14 in vitro into fatty acids and cholesterol in the liver of pregnant rats has been reported. In view of the scattered information on the regulation of placental and liver metabolism in pregnancy, a study was undertaken of the behaviour of enzymes of glycolysis, gluconeogenesis and lipogenesis, known of their adaptive susceptibility to hormonal control. Since the placenta undergoes extensive functional and morphological changes with the progress of pregnancy, the factor of placental age had to be taken into account in the evaluation of effects on enzyme activity. This report presents observations on the influence of length of gestation on the activity of several placental enzymes of glucose and lipid metabolism in comparison to the activity of analogous enzymes in the liver of the same pregnant rats. MATERIALS AND METHODS
Pregnant albino rats of Hebrew University strain, weighing 15o-2oo g, and nonpregnant control female rats were fed ad libitum a pelleted chow containing 60% carbohydrate, 20 ~o protein, 5 % fat and the rest salts and non-utilizable residue. The rats were anesthetized by a short exposure to ether. After the collection of blood from the abdominal aorta, hysterectomy was performed. The liver and placental tissues were removed and homogenized (1:3, w/v) in 0.20 M sucrose solution containing 20 mM triethanolamine (pH 7.4), I mM disodium EDTA and I mM dithioerythritol. The homogenates were centrifuged at IOOOOO×g at 4 °C for 45 min. The supernatant fluids were used for enzyme assays. E n z y m e assays
Pyruvate kinase (EC 2.7.1.4o ) activity was measured according to Bticher and Pfleiderer 15. Alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1) activities were measured as described by Bergmeyer and Bernt 1.. Glucose-6-phosphate dehydrogenase (EC I. i. 1.49), 6-phosphogluconate dehydrogenase (EC I.I.I.43), ATP citrate lyase (EC 4.I.3.8), NADP-malate dehydrogenase (EC 1.1.1.4o ) and acetyl-CoA carboxylase (EC 6.4.1.2) activities were measured as outlined in a previous publicationlL The activity of the latter enzyme was determined after maximal pre-activation in the presence of citrate. Phosphoenolpyruvate carboxylase (EC 4.1.1.32) activity was determined by a Bioahim. Biophys. Acta, 279 (1972) 424-43°
426
Y. Z. DIAMANT, E. SHAFRIR
modification of the method of Chang and Lane is, based on the rate of enzyme-catalyzed exchange between KH14COs and unlabeled oxaloacetate. The reaction mixture in a final volume of 0. 5 ml comprised (in/~moles) imidazole buffer (pH 6.6), IOO ; ITP, 1.5 ; MnCl~, 1.25 ; dithioerythritol, I ; sodium oxaloacetate freshly prepared and neutralized, 2; KH14CO3, 15 (I/~Ci). In the case of liver, 5-1o pl and in the case of placenta a quantity of IOO #1 of the supernatant fluid was used to obtain radioactivity incorporation exceeding that of blank by a factor of IO at least. The reaction was stopped at 2 and 4 min of incubation at 37 °C (the linearity of the reaction was established for at least 5 rain) by the addition of 0.5 ml of a solution containing 15 Fmoles disodium EDTA, 2.5 #moles N A D H and 1. 5 units of NAD-malate dehydrogenase, in order to convert the oxaloacetate to malate. After an additional 5 rain of incubation, 0.5 ml of lO% trichloroacetic acid was added, the CO~ expelled by gassing with a CO2-O2 mixture and the acid-stable radioactivity remaining in the reaction mixture determined. Since the rate of exchange between carbonate and oxaloacetate exceeds the rate of catalytic phosphorylation of oxaloacetate to phosphoenolpyruvate by the factor of 4 (ref. I8), it provides a sensitive measure for the determination of low enzyme activity. All enzyme activities were determined at 37 °C and expressed as nmoles of the substrate metabolized per min per mg protein 19 in the respective tissue supernatant fraction. RESULTS
Enzyme activity values in the placenta and liver and the changes occurring in the progress of gestation are summarized in Tables I and II. Firstly, the activities of placental and liver enzymes are compared on the i5th day of gestation. Pyruvate kinase, an important regulatory enzyme of glycolysis, was found to have a very marked activity in the placental tissue (Table I), about four times higher than in the liver of the same pregnant rats (Table II). There was no significant difference in liver pyruvate kinase activity between pregnant and nonpregnant rats. Phosphoenolpyruvate carboxyl~e, one of the rate-limiting enzymes of gluconeogenesis, had a much lower activity in the placenta (Table I), as compared with the activity of this enzyme in the liver of either pregnant or nonpregnant rats (Table II). The activity of alanine and aspartate aminotransferases was also low in the placenta as compared to the liver. Pregnancy b y itself did not appear to affect the activity of these liver enzymes. Of the enzymes contributing reduced pyridine trinucleotides to lipid synthesis, NADP-malate dehydrogenase activity in placental tissue was 30-50% of that in the liver of pregnant or nonpregnant rats. I t is noteworthy that the activity of this enzyme in the liver significantly rose as a result of pregnancy. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in placental tissue amounted only to a fraction of the activity of these enzymes in the liver. Also in this case a significant rise in enzyme activity in the liver was apparent as a result of pregnancy. ATP citrate lyase showed a relatively high activity in the placenta, close to Biochim. Biophys. Acta, 279 (1972) 424-43 o
O
I
~D
~D
TABLE I
2488 ± lO4 4.5 4- 0.6 i 6 3 8 4- 173" 3.7 + 0.8 615 4- 68* 2.1 4- o.2*
Alanine aminotransferase
Glucose-6phosphate dehydrogenase
i o . i ± 1.6 6.8 4- 0.9 35.9 4- 4.3 6.9 4- 1.2 3-3 4- o.7* 31.6 ! 4.4 6.0 + i . o * 1. 7 4- o.3* 25. 7 zk 3.3
Phosphoenol- Aspartate pyruvate aminocarboxylase transferase 51.3 4- 4 .0 32.5 ± 2.4 25.3 4- 3-7*
6-Phosphogluconate dehydrogenase 33.3 4- 6.4 16. 3 4- 1.2" lO.6 ~- o.5*
NADPmalate dehydrogenase
218 ~ 23
30o±
23
155 t
io
122 4- 15
274 4- 15
35 ° 4- IO 3 o 2 4 - 17 292 4- 8*
6-Phosphogluconate dehydrogenase
6 6 . 6 4 - 2.6
96,4 ~ 7.3 5o.o4-6.4* 51.3 ± 3.4*
NADPmalate dehydrogenase
* S i g n i f i c a n t difference f r om t h e v a l u e a t I 5 t h d a y of p r e g n a n c y a t l e v e l of P < 0.02 a t least.
736 ~ 52
289 4- 21 167 4- 22 184 4- 23 241 4 - 2 2 1 3 o 4 - 13 1 7 5 4 - IO 392 4- 24* 88 ± IO* 152 ± 17
165 4- 25 189 4- 20 226 4- 19
15 18 20 Nonpregnant
628 ± 32 563 4- 2o 6o2 4- 3 °
Phosphoenol- Aspartate Alanine Glucose-6 pyruvate aminoaminophosphate carboxylase transferase transferase dehydrogenase
Day of Pyruvate pregnancy kinase
26. 7 ± 1.6
28.r 4- 3.2 34.5 ± 3 .1 37.8 ± 4.6
24.2 4- 3 ,6 12.4±2.o* 7.2 ~ 1.2" 21.5 ± 3.3
A cetylCoA carboxylase
A TP citrate lyase
73.1 4- 1.4
6 9 . 0 4- I . o 73.0 4- 0.6 74.0 4- I , I
Cytoplasmic protein content (mg/g wet wt)
32.5 4- 1.3 33.2 4- o.5 35.o -h o.9
AcetylCytoplasmic CoA protein carboxylase content (mg[g wet wt)
lO.8 4- 1.2 0.92 i o . i o 9.5 ± i . o 1.26 4- o.3o 5.2 4- 1.5" o.83 4- o . i o
ATPcitrate lyase
V a l u e s in t h e t a b l e a r e m e a n s 4- S.E. for 9 - 1 3 r a t s i n e a c h group. A c t i v i t i e s a re e x p r e s s e d as n m o l e s / m i n p e r m g p r o t e i n .
CHANGES IN THE ACTIVITY OF LIVER ENZYMES DURING PREGNANCY
TABLE II
* S i g n i f i c a n t difference f r om t h e v a l u e a t I 5 t h d a y of p r e g n a n c y a t t h e l e ve l of P < 0.02 a t least.
15 18 2o
Day of Pyruvate pregnancy kinase
CHANGES IN THE ACTIVITY OF PLACENTAL ENZYMES DURING PREGNANCY V a lue s in t h e t a b l e ar e m e a n s -k S.E. for 9 - 1 3 r a t s in e a c h group. A c t i v i t i e s are e x p r e s s e d as n m o l e s / m i n p e r m g p r o t e i n .
to
428
Y. Z. DIAMANT, E. SHAFRIR
5o~o of that found in the liver. The small increase in the activity of this enzyme in the liver of pregnant as compared to nonpregnant rats, was not significant. The activity of acetyl-CoA carboxylase, the rate-limiting enzyme in the pathway of fatty acid synthesis, was very low in placental tissue. It amounted to only about 3~o of that found in the liver of pregnant or nonpregnant rats (Table II). Next, the changes in enzyme activity, which occurred during the last 5 days of gestation are compared. A general fall in the activity of most enzymes was observed in the placenta despite virtually unchanged tissue protein content (Table I). The activity of pyruvate kinase fell on Day 2o of pregnancy to a value as low as 1/4 of that on Day 15 of pregnancy. Of the two placental aminotransferases, particularly the activity of alanine aminotransferase fell on Day 2o to 1/4 of its value on Day 15. The activity of phosphoenolpyruvate carboxylase also declined with the progress of gestation. The activity of NADPH-generating enzymes in the placenta, 6-phosphogluconate dehydrogenase and NADP-malate dehydrogenase fell to 1/2 and I/3 of its initial values, respectively. The activity of glucose-6-phosphate dehydrogenase remained without appreciable change. Of the enzymes directly related to lipogenesis, the activity of ATP citrate lyase declined particularly between Days 18 and 2o of the gestation, whereas that of acetyl-CoA carboxylase remained without significant alteration. The effects of advancing gestation on liver enzyme activity was much less marked (Table II). Pyruvate kinase and phosphoenolpyruvate carboxylase activities remained practically unchanged; the activity of aspartate aminotransferase tended to rise, whereas that of alanine aminotransferase was reduced by 5o}~. There was a trend towards decrease in the activity of NADPH-generating enzymes. ATP citrate lyase decreased most markedly of all liver enzymes. At Day 2o its activity was 1/3 that at Day 15. On the other hand, acetyl-CoA carboxylase activity showed a slight, though insignificant, increase in activity. DISCUSSION
The presence of a highly active glycolytic pathway in rat placenta is indicated by the remarkably high activity of pyruvate kinase. The significant decrease in activity of this enzyme during the last 5 days of pregnancy suggests a decrease in glucose utilization. This is also evident from the decrease in the activity of the pentose shunt pathway close to term. These findings are in agreement with the data on oxidation of EI-l*Clglucose and I6-1*C?glucose to 14CO~ in slices of human placental tissue, which indicated a significant activity of the shunt in early and midterm placentas and a marked decrease in activity at term2,a, 20. The possibility of gluconeogenesis in the placenta is suggested by the demonstration of phosphoenolpyruvate carboxylase activity, one of the rate-limiting enzymes of the gluconeogenic pathway. However, it does not appear to be of great importance since the activity of this enzyme is rather low. The low activity of alanine and aspartate aminotransferases, which are instrumental in the supply of glucogenic precursors, lends support to this conclusion. It is known that the placenta can synthesize cholesterol, which may serve as a precursor of steroid hormones such as pregnenolone 21 and progesterone 22 which in Biochim. Biophys. Acta, 279 (1972) 424-43o
E N Z Y M E S I N P L A C E N T A A N D L I V E R OF P R E G N A N T RATS
429
turn may be converted to other steroid hormones23,~4. It is pertinent in this respect that the activities of placental NADP-malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as ATP citrate lyase markedly exceeded the activity of acetyl-CoA carboxylase. In the liver the activity of these enzymes was of a magnitude similar to that of acetyl-CoA carboxylase. The question arises whether the activity of placental enzymes generating NADPH or promoting acetyl-CoA transport through citrate cleavage is linked necessarily to the synthesis of fatty acids or of other lipids, possibly sterols. The low activity of acetyl-CoA carboxylase suggests that de novo fatty acid synthesis is of minor importance in the placenta. With the progress of pregnancy, a substantial decrease in the activity of both placental and liver NADP-malate dehydrogenase was observed. Smaller decreases were also seen in the activity of enzymes of the pentose shunt pathway. Walker et al. ~ also reported a decrease in glucose-6-phosphate dehydrogenase activity in rat placenta toward term, although this was not significant in our hands. According to Betz and WarrenL Marks and Banks ~°, Levy2L and Tepperman et al. 2e, the decline in activity of these enzymes may be related to the rising levels of steroid hormones in pregnancy, such as estrogen and dehydroepiandrosterone. No changes in liver pyruvate kinase activity were induced by the gestation itself or during its progress. The results of HagermanlL showing and increased rate of glucose utilization by slices of pregnant rat liver, will have to be reconciled with this observation. The decrease in the activity of liver alanine aminotransferase during pregnancy is compatible with the earlier findings, that less pyruvate is converted to glucose by the liver of pregnant as compared to nonpregnant rats in vitrolL or in vivo ~. However, no conclusion may be reached as to the total glucogenic capacity, since the activity of aspartate aminotransferase tended to increase and the activity of phosphoenolpyruvate carboxylase remained without appreciable change with advancing pregnancy. When the gestation-induced enzyme activity changes in the liver and placenta are compared, no uniform pattern is apparent. The NADPH-generating enzymes and alanine aminotransferase decreased in both tissues; pyruvate kinase decreased only in the placenta and changes in aspartate aminotransferase were in opposite directions in the two tissues. This calls for further studies of the hormonal set-up peculiar to gestation, which induces these selective changes in the activity of placental and hepatic enzymes. ACKNOWLEDGEMENTS
We are grateful to Prof. W. Z. Polishuk of the Obstetrics and Gynecology Department, Hadassah-University Hospital, for his interest in the conduct of this investigation. We are indebted to Mrs A. Gal for her excellent assistance in enzyme activity measurements. This work was supported by Research Grant No. M70.72 C from Population Council, New York, U.S.A.
Biochim. Biophys. Aaa, 279 (1972) 424-43o
43 °
Y. Z. DIAMANT, E. SHAFRIR
REFERENCES I A. St. G. H u g g e r a n d S. A. Morrison, J. Physiol., 129 (1955) 68P. 2 J. G i n s b u r g a n d M. K. Jeacock, Biochim. Biophys. Acta, 90 (1964) 166. 3 D. G. Walker, M. A. Lea, G. R o s s i t e r a n d M. E. B. Addison, Arch. Biochem. Biophys., 12o (1967) 646. 4 G. Betz a n d Y. C. W a r r e n , Acta Endocrinol., 49 (1965) 475 C. A. Villee, J. Biol. Chem., 2o 5 (1953) I13. 6 J. E. Carter a n d G. W e b e r , Am. J. Obstet. Gynecol., 95 (1966) 914. 7 D. L. DiPietro, J. Gutierrez-Correa a n d J. H. T h a i d i g s m a n , Biochem. J., lO 3 (1967) 246. 8 M. K. Y o u n o s z a i a n d J. C. H o w o r t h , Am. J. Obstet. Gynecol., lO 3 (1968) 262. 9 J. F. R o u x , A. Grigorian a n d Y. T a k e d a , Nature, 216 (1967) 819. IO U. Kleine, Clin. Chim. Acta, 17 (1967) 95. I I N. H o s o y a , D. H a g e r m a n a n d C. A. Villee, Bioehem. J., 76 (196o) 297. I2 D. A. H a g e r m a n , Endocrinology, 7 ° (1962) 88. 13 E. Herrera, R. H. K n o p p a n d N. Freinkel, J. Clin. Invest., 48 (1969) 2260. 14 W. N. D a n n e n b u r g , R. L. B u t t a n d N. H. Leake, Proc. Soc. Exp. Biol. Med., 115 (1964) 504 . 15 T. Bflcher a n d G. Pfleiderer, in S. P. Colowick a n d N. O. K a p l a n , Methods in Enzymology, A c a d e m i c Press, N e w York, 1955, p. 435. 16 H. U. B e r g m e y e r a n d E. B e r n t , in H. U. B e r g m e y e r , Methods of Enzymatic Analysis, A c a d e m i c Press, New Y o r k a n d L o n d o n , 1963, pp. 837, 846. 17 S. D i a m a n t , E. Gorin a n d E. Shafrir, Eur. J. Biochem., 26 (1972) 553. 18 H. C. C h a n g a n d M. D. Lane, J. Biol. Chem., 241 (1966) 2413 . 19 O. H. Lowry, N. J. R o s e b r o u g h , A. L. F a r r a n d R. J. R a n d a l l , J. Biol. Chem., 193 (1951) 256. 2o P. A. M a r k s a n d J. B a n k s , Proc. Natl. Acad. Sci., U.S., 46 (196o) 447. 21 S. Solomon, J. Clin. Endocrinol. Metab., 26 (1966) 762. 22 G. Morrison, R. A. Meigs a n d K. J. R y a n , Steroids, 6, Suppl. 2 (1965) 177. 23 E. Diczfalusy a n d P. Toren, Vitam. Horm., 19 (1961) 229. 24 S. S o l o m o n a n d H. G. Freisen, Annu. Rev. Med., 19 (1968) 299. 25 H. R. L e v y , Biochem. Biophys. Res. Commun., 6 (1961) 49. 26 H. M. T e p p e r m a n , S. A. De L a G a r z a a n d J. T e p p e r m a n , Am. J. Physiol., 214 (1968) 1126.
Biochim. Biophys. Acta, 279 (1972) 424-43 °
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 26952
ENZYMES OF CARBOHYDRATE AND L I P I D METABOLISM IN T H E PLACENTA AND LIVER OF PREGNANT RATS
Y O R A M Z. D I A M A N T AND E L E A Z A R S H A F R I R
Departments of Obstetrics and Gynecology and Clinical Biochemistry, Hadassa University Hospital and Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Received J u l y 2oth, 1972)
SUMMARY
Changes in the activity of enzymes involved in glycolysis, gluconeogenesis and lipogenesis during the last days of gestation were determined in the placenta and liver of pregnant rats and expressed in relation to the cytoplasmic protein content of these tissues. On Day 15 of gestation the activity of pyruvate kinase (EC 2.7.1.4o) in the placenta was considerably higher than in the liver, whereas the activity of placental enzymes associated with gluconeogenesis was much lower than in the liver. This indicated that the placental capacity of glucose utilization is preponderant over gluconeogenesis. The activity of enzymes associated with lipid synthesis was lower in the placenta in comparison to the liver. However, the activity of enzymes of NADPH generation and of ATP citrate lyase (EC 4.1.3.8) in relation to that of acetyl-CoA carboxylase (EC 6.4.1.2 ) was markedly higher in the placenta than in the liver. This was suggestive of the importance of synthesis of lipids other than fatty acids in the placenta. With the progress of gestation a decrease in the activity of most placental enzymes was observed, particularly in that of pyruvate kinase. In the liver the activity of enzymes of NADPH generation and of ATP citrate lyase also decreased. The possible background of these changes in enzyme activity in the liver and placenta is discussed.
INTRODUCTION
The metabolism of carbohydrates and lipids in the placenta provides the requirements of energy for the synthesis of a range of sterols and steroid hormones, the active transport of metabolites and other functions which are assumed to be auxilliary to the fetal liver. Enzymatic pathways are known to be present for the conversion of glucose to glycogen or its degradation in the glycolytic pathway and in the hexose monophosphate shunt 1-5. Significant production of glucose from pyruvate was observed in human placenta in the early stage of gestation 5. The decline in placental glucose-6-phosBiochim. Biophys. Acta, 279 (1972) 424-43 o
E N Z Y M E S IN P L A C E N T A A N D L I V E R O F P R E G N A N T R A T S
425
phatase activity with gestationS, e was considered to explain the absence of significant gluconeogenesis at term. On the other hand, the small rate of glucose and fructose production from labeled pyruvate was found not to differ in the young or term human placentasL It was attributed to nonspecific hexose phosphate hydrolysis by the abundant placental alkaline phosphatase~,L The quantity of lipid in the placenta is small s. Placental triglycerides and phospholipids are formed from esterification of fatty acids either taken up from maternal blood or synthesized de novo% 1°. Basic enzymatic steps of this pathway have been investigated in the placenta by Hosoya et al. ~. With regard to hepatic carbohydrate and fat metabolism in pregnancy, an enhancement in glycolysis with a concomitant reduction in oxidative metabolism was suggested 12. No changes in liver gluconeogenesis or glycogen synthesis have been observed in pregnant fed animals as compared to normal controls t3. An increased incorporation of I2-14CJpyruvate ~ and EI-14C]acetate14 in vitro into fatty acids and cholesterol in the liver of pregnant rats has been reported. In view of the scattered information on the regulation of placental and liver metabolism in pregnancy, a study was undertaken of the behaviour of enzymes of glycolysis, gluconeogenesis and lipogenesis, known of their adaptive susceptibility to hormonal control. Since the placenta undergoes extensive functional and morphological changes with the progress of pregnancy, the factor of placental age had to be taken into account in the evaluation of effects on enzyme activity. This report presents observations on the influence of length of gestation on the activity of several placental enzymes of glucose and lipid metabolism in comparison to the activity of analogous enzymes in the liver of the same pregnant rats. MATERIALS AND METHODS
Pregnant albino rats of Hebrew University strain, weighing 15o-2oo g, and nonpregnant control female rats were fed ad libitum a pelleted chow containing 60% carbohydrate, 20 ~o protein, 5 % fat and the rest salts and non-utilizable residue. The rats were anesthetized by a short exposure to ether. After the collection of blood from the abdominal aorta, hysterectomy was performed. The liver and placental tissues were removed and homogenized (1:3, w/v) in 0.20 M sucrose solution containing 20 mM triethanolamine (pH 7.4), I mM disodium EDTA and I mM dithioerythritol. The homogenates were centrifuged at IOOOOO×g at 4 °C for 45 min. The supernatant fluids were used for enzyme assays. E n z y m e assays
Pyruvate kinase (EC 2.7.1.4o ) activity was measured according to Bticher and Pfleiderer 15. Alanine aminotransferase (EC 2.6.1.2) and aspartate aminotransferase (EC 2.6.1.1) activities were measured as described by Bergmeyer and Bernt 1.. Glucose-6-phosphate dehydrogenase (EC I. i. 1.49), 6-phosphogluconate dehydrogenase (EC I.I.I.43), ATP citrate lyase (EC 4.I.3.8), NADP-malate dehydrogenase (EC 1.1.1.4o ) and acetyl-CoA carboxylase (EC 6.4.1.2) activities were measured as outlined in a previous publicationlL The activity of the latter enzyme was determined after maximal pre-activation in the presence of citrate. Phosphoenolpyruvate carboxylase (EC 4.1.1.32) activity was determined by a Bioahim. Biophys. Acta, 279 (1972) 424-43°
426
Y. Z. DIAMANT, E. SHAFRIR
modification of the method of Chang and Lane is, based on the rate of enzyme-catalyzed exchange between KH14COs and unlabeled oxaloacetate. The reaction mixture in a final volume of 0. 5 ml comprised (in/~moles) imidazole buffer (pH 6.6), IOO ; ITP, 1.5 ; MnCl~, 1.25 ; dithioerythritol, I ; sodium oxaloacetate freshly prepared and neutralized, 2; KH14CO3, 15 (I/~Ci). In the case of liver, 5-1o pl and in the case of placenta a quantity of IOO #1 of the supernatant fluid was used to obtain radioactivity incorporation exceeding that of blank by a factor of IO at least. The reaction was stopped at 2 and 4 min of incubation at 37 °C (the linearity of the reaction was established for at least 5 rain) by the addition of 0.5 ml of a solution containing 15 Fmoles disodium EDTA, 2.5 #moles N A D H and 1. 5 units of NAD-malate dehydrogenase, in order to convert the oxaloacetate to malate. After an additional 5 rain of incubation, 0.5 ml of lO% trichloroacetic acid was added, the CO~ expelled by gassing with a CO2-O2 mixture and the acid-stable radioactivity remaining in the reaction mixture determined. Since the rate of exchange between carbonate and oxaloacetate exceeds the rate of catalytic phosphorylation of oxaloacetate to phosphoenolpyruvate by the factor of 4 (ref. I8), it provides a sensitive measure for the determination of low enzyme activity. All enzyme activities were determined at 37 °C and expressed as nmoles of the substrate metabolized per min per mg protein 19 in the respective tissue supernatant fraction. RESULTS
Enzyme activity values in the placenta and liver and the changes occurring in the progress of gestation are summarized in Tables I and II. Firstly, the activities of placental and liver enzymes are compared on the i5th day of gestation. Pyruvate kinase, an important regulatory enzyme of glycolysis, was found to have a very marked activity in the placental tissue (Table I), about four times higher than in the liver of the same pregnant rats (Table II). There was no significant difference in liver pyruvate kinase activity between pregnant and nonpregnant rats. Phosphoenolpyruvate carboxyl~e, one of the rate-limiting enzymes of gluconeogenesis, had a much lower activity in the placenta (Table I), as compared with the activity of this enzyme in the liver of either pregnant or nonpregnant rats (Table II). The activity of alanine and aspartate aminotransferases was also low in the placenta as compared to the liver. Pregnancy b y itself did not appear to affect the activity of these liver enzymes. Of the enzymes contributing reduced pyridine trinucleotides to lipid synthesis, NADP-malate dehydrogenase activity in placental tissue was 30-50% of that in the liver of pregnant or nonpregnant rats. I t is noteworthy that the activity of this enzyme in the liver significantly rose as a result of pregnancy. The activity of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in placental tissue amounted only to a fraction of the activity of these enzymes in the liver. Also in this case a significant rise in enzyme activity in the liver was apparent as a result of pregnancy. ATP citrate lyase showed a relatively high activity in the placenta, close to Biochim. Biophys. Acta, 279 (1972) 424-43 o
O
I
~D
~D
TABLE I
2488 ± lO4 4.5 4- 0.6 i 6 3 8 4- 173" 3.7 + 0.8 615 4- 68* 2.1 4- o.2*
Alanine aminotransferase
Glucose-6phosphate dehydrogenase
i o . i ± 1.6 6.8 4- 0.9 35.9 4- 4.3 6.9 4- 1.2 3-3 4- o.7* 31.6 ! 4.4 6.0 + i . o * 1. 7 4- o.3* 25. 7 zk 3.3
Phosphoenol- Aspartate pyruvate aminocarboxylase transferase 51.3 4- 4 .0 32.5 ± 2.4 25.3 4- 3-7*
6-Phosphogluconate dehydrogenase 33.3 4- 6.4 16. 3 4- 1.2" lO.6 ~- o.5*
NADPmalate dehydrogenase
218 ~ 23
30o±
23
155 t
io
122 4- 15
274 4- 15
35 ° 4- IO 3 o 2 4 - 17 292 4- 8*
6-Phosphogluconate dehydrogenase
6 6 . 6 4 - 2.6
96,4 ~ 7.3 5o.o4-6.4* 51.3 ± 3.4*
NADPmalate dehydrogenase
* S i g n i f i c a n t difference f r om t h e v a l u e a t I 5 t h d a y of p r e g n a n c y a t l e v e l of P < 0.02 a t least.
736 ~ 52
289 4- 21 167 4- 22 184 4- 23 241 4 - 2 2 1 3 o 4 - 13 1 7 5 4 - IO 392 4- 24* 88 ± IO* 152 ± 17
165 4- 25 189 4- 20 226 4- 19
15 18 20 Nonpregnant
628 ± 32 563 4- 2o 6o2 4- 3 °
Phosphoenol- Aspartate Alanine Glucose-6 pyruvate aminoaminophosphate carboxylase transferase transferase dehydrogenase
Day of Pyruvate pregnancy kinase
26. 7 ± 1.6
28.r 4- 3.2 34.5 ± 3 .1 37.8 ± 4.6
24.2 4- 3 ,6 12.4±2.o* 7.2 ~ 1.2" 21.5 ± 3.3
A cetylCoA carboxylase
A TP citrate lyase
73.1 4- 1.4
6 9 . 0 4- I . o 73.0 4- 0.6 74.0 4- I , I
Cytoplasmic protein content (mg/g wet wt)
32.5 4- 1.3 33.2 4- o.5 35.o -h o.9
AcetylCytoplasmic CoA protein carboxylase content (mg[g wet wt)
lO.8 4- 1.2 0.92 i o . i o 9.5 ± i . o 1.26 4- o.3o 5.2 4- 1.5" o.83 4- o . i o
ATPcitrate lyase
V a l u e s in t h e t a b l e a r e m e a n s 4- S.E. for 9 - 1 3 r a t s i n e a c h group. A c t i v i t i e s a re e x p r e s s e d as n m o l e s / m i n p e r m g p r o t e i n .
CHANGES IN THE ACTIVITY OF LIVER ENZYMES DURING PREGNANCY
TABLE II
* S i g n i f i c a n t difference f r om t h e v a l u e a t I 5 t h d a y of p r e g n a n c y a t t h e l e ve l of P < 0.02 a t least.
15 18 2o
Day of Pyruvate pregnancy kinase
CHANGES IN THE ACTIVITY OF PLACENTAL ENZYMES DURING PREGNANCY V a lue s in t h e t a b l e ar e m e a n s -k S.E. for 9 - 1 3 r a t s in e a c h group. A c t i v i t i e s are e x p r e s s e d as n m o l e s / m i n p e r m g p r o t e i n .
to
428
Y. Z. DIAMANT, E. SHAFRIR
5o~o of that found in the liver. The small increase in the activity of this enzyme in the liver of pregnant as compared to nonpregnant rats, was not significant. The activity of acetyl-CoA carboxylase, the rate-limiting enzyme in the pathway of fatty acid synthesis, was very low in placental tissue. It amounted to only about 3~o of that found in the liver of pregnant or nonpregnant rats (Table II). Next, the changes in enzyme activity, which occurred during the last 5 days of gestation are compared. A general fall in the activity of most enzymes was observed in the placenta despite virtually unchanged tissue protein content (Table I). The activity of pyruvate kinase fell on Day 2o of pregnancy to a value as low as 1/4 of that on Day 15 of pregnancy. Of the two placental aminotransferases, particularly the activity of alanine aminotransferase fell on Day 2o to 1/4 of its value on Day 15. The activity of phosphoenolpyruvate carboxylase also declined with the progress of gestation. The activity of NADPH-generating enzymes in the placenta, 6-phosphogluconate dehydrogenase and NADP-malate dehydrogenase fell to 1/2 and I/3 of its initial values, respectively. The activity of glucose-6-phosphate dehydrogenase remained without appreciable change. Of the enzymes directly related to lipogenesis, the activity of ATP citrate lyase declined particularly between Days 18 and 2o of the gestation, whereas that of acetyl-CoA carboxylase remained without significant alteration. The effects of advancing gestation on liver enzyme activity was much less marked (Table II). Pyruvate kinase and phosphoenolpyruvate carboxylase activities remained practically unchanged; the activity of aspartate aminotransferase tended to rise, whereas that of alanine aminotransferase was reduced by 5o}~. There was a trend towards decrease in the activity of NADPH-generating enzymes. ATP citrate lyase decreased most markedly of all liver enzymes. At Day 2o its activity was 1/3 that at Day 15. On the other hand, acetyl-CoA carboxylase activity showed a slight, though insignificant, increase in activity. DISCUSSION
The presence of a highly active glycolytic pathway in rat placenta is indicated by the remarkably high activity of pyruvate kinase. The significant decrease in activity of this enzyme during the last 5 days of pregnancy suggests a decrease in glucose utilization. This is also evident from the decrease in the activity of the pentose shunt pathway close to term. These findings are in agreement with the data on oxidation of EI-l*Clglucose and I6-1*C?glucose to 14CO~ in slices of human placental tissue, which indicated a significant activity of the shunt in early and midterm placentas and a marked decrease in activity at term2,a, 20. The possibility of gluconeogenesis in the placenta is suggested by the demonstration of phosphoenolpyruvate carboxylase activity, one of the rate-limiting enzymes of the gluconeogenic pathway. However, it does not appear to be of great importance since the activity of this enzyme is rather low. The low activity of alanine and aspartate aminotransferases, which are instrumental in the supply of glucogenic precursors, lends support to this conclusion. It is known that the placenta can synthesize cholesterol, which may serve as a precursor of steroid hormones such as pregnenolone 21 and progesterone 22 which in Biochim. Biophys. Acta, 279 (1972) 424-43o
E N Z Y M E S I N P L A C E N T A A N D L I V E R OF P R E G N A N T RATS
429
turn may be converted to other steroid hormones23,~4. It is pertinent in this respect that the activities of placental NADP-malate dehydrogenase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase as well as ATP citrate lyase markedly exceeded the activity of acetyl-CoA carboxylase. In the liver the activity of these enzymes was of a magnitude similar to that of acetyl-CoA carboxylase. The question arises whether the activity of placental enzymes generating NADPH or promoting acetyl-CoA transport through citrate cleavage is linked necessarily to the synthesis of fatty acids or of other lipids, possibly sterols. The low activity of acetyl-CoA carboxylase suggests that de novo fatty acid synthesis is of minor importance in the placenta. With the progress of pregnancy, a substantial decrease in the activity of both placental and liver NADP-malate dehydrogenase was observed. Smaller decreases were also seen in the activity of enzymes of the pentose shunt pathway. Walker et al. ~ also reported a decrease in glucose-6-phosphate dehydrogenase activity in rat placenta toward term, although this was not significant in our hands. According to Betz and WarrenL Marks and Banks ~°, Levy2L and Tepperman et al. 2e, the decline in activity of these enzymes may be related to the rising levels of steroid hormones in pregnancy, such as estrogen and dehydroepiandrosterone. No changes in liver pyruvate kinase activity were induced by the gestation itself or during its progress. The results of HagermanlL showing and increased rate of glucose utilization by slices of pregnant rat liver, will have to be reconciled with this observation. The decrease in the activity of liver alanine aminotransferase during pregnancy is compatible with the earlier findings, that less pyruvate is converted to glucose by the liver of pregnant as compared to nonpregnant rats in vitrolL or in vivo ~. However, no conclusion may be reached as to the total glucogenic capacity, since the activity of aspartate aminotransferase tended to increase and the activity of phosphoenolpyruvate carboxylase remained without appreciable change with advancing pregnancy. When the gestation-induced enzyme activity changes in the liver and placenta are compared, no uniform pattern is apparent. The NADPH-generating enzymes and alanine aminotransferase decreased in both tissues; pyruvate kinase decreased only in the placenta and changes in aspartate aminotransferase were in opposite directions in the two tissues. This calls for further studies of the hormonal set-up peculiar to gestation, which induces these selective changes in the activity of placental and hepatic enzymes. ACKNOWLEDGEMENTS
We are grateful to Prof. W. Z. Polishuk of the Obstetrics and Gynecology Department, Hadassah-University Hospital, for his interest in the conduct of this investigation. We are indebted to Mrs A. Gal for her excellent assistance in enzyme activity measurements. This work was supported by Research Grant No. M70.72 C from Population Council, New York, U.S.A.
Biochim. Biophys. Aaa, 279 (1972) 424-43o
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Y. Z. DIAMANT, E. SHAFRIR
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