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Effects of progesterone on some enzymes of fat and carbohydrate metabolism in rat liver
Effects of progesterone on some enzymes of fat and carbohydrate metabolism in rat liver CHARLES
H.
JUNNOSUKE MAX
DAHM,
JR.,
M.D.
MINAGAWA,
JELLINEK,
M.D.
PH.D.
St. Louis. Missouri The known effect of progesterone on carbohydrate metabolism prompted a study of some of the hepatic “lipogenic” and “gluconeogenic” enzymes in rats treated with progesterone. Several enzymes providing lipid precursors (phosphofructokinase, malic enzyme, glucose-6-phosphate dehydrogenase, and citrate cleavage enzyme) showed increased specific activity. These changes may represent insulin effects. Specific activity of phosphoenolpyruvate carboxykinase, usually associated with control of gluconeogenesis, was also increased. The latter is compatible with increased capability for glycogenesis, which is recognized as a progesterone effect. (AM. J. OBSTET. GYNECOL. 129: 130, 1977.)
taneously. for 14 days, followed by 2.5 mg. twice daily for seven days. The animals were killed after 2 1 days of treatment. Sodium pentobarbital was given intraperitoneally in a dose of 40 mg. per kilogram. After anesthesia was induced, the livers were excised, blotted free of blood, and placed on ice. A 10 per cent w/v liver homogenate was prepared in cold 30 per cent sucrose solution in a Dual1 homogenizer. The soluble fraction was then separated by ultracentrifugation at 40,000 r.p.m. for one hour. The resultant pellet was resuspended in 12 ml. of cold sucrose solution, and cellular debris was removed by centrifugation at 8,000 r.p.m. for 10 minutes. The supernatant containing the mitochondria was then recentrifuged at 25,000 r.p.m. for 30 minutes. The mitochondrial pellet was resuspended in 5 ml. of 30 per cent sucrose solution and the mitochondrial membrane was disrupted by sonication for approximately one minute. Enzymes studied included phosphofructokinase (PFK), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), glycerol-3-phosphate dehydrogenase (G-3-PD), citrate cleavage enzyme (CCE), fatty acid synthetase (FAS). pyruvate carboxylase (PC). phosphoenolpyruvate carboxykinase (PEPCK), fructose- 1,6-diphosphatase (FDPase), and lactate dehydrogenase (LDH). PC was measured by the method of Ballard and Hanson3 PEPCK was measured by the method of Chang and Lane,4 and FDPase was measured by the method of Carlson and associates.5 CCE was measured by the method of Inoue and associateQ while FAS was determined by the method of Hsu and
that progeSterOne influences the intermediary metabolism of carbohydrate in the liver. Matute and Kalkhoff’ have shown that progesterone, given singly or in combination with estradiol, suppresses the incorporation of alanineU”‘C and pyruvate-3-i% into glucose. However, hepatic glycogen deposition is increased. Sladek’ has shown that, while gluconeogenesis from alanine is inhibited by estradiol administration, incorporation of alanine into glycogen is increased. The present investigation was undertaken to study the effects of progesterone on some of the hepatic enzymes associated with lipogenesis and gluconeogenesis in rats. IT
IS
GENERALLY
ACCEPTED
Methods Intact female Wistar rats weighing approximately 200 grams were used. Twenty-four animals were studied in all determinations. Food and water were provided freely throughout the course of the treatment. Progesterone (12.5 mg. per milliliter of sesame oil) was given in a dose of 1.25 mg. twice daily, subcuFrom the Departments of Obstetrics-Gynecology Surgery, St. Louis University, and St. Louis Hospital. Supported in part No. HLO-6312-15. Received
for
publication
Accepted April Reprint
by National March
Institutes
and City
of Health
Grant
4, 1977.
14. 1977.
requests: Dr. Charles
of Obstetrics
and
Louis University, Missouri 63104.
H. Dahm, Jr., Department School of Medicine, St. 132.5 S. Grand Blvd., St. Louis,
Gynecology,
130
Volume Number
129 2
Progesterone effects on metabolism
Control
cl
Progesterone
m
p
‘P <0.005 “P io.COo5
2
.cl
q
Control
131
‘P
Progesterone
PC
Fig. 1. Effect of progesterone “lipogenic” enzymes: PFK, ME, FAS.
on some “glycolytic” G-6-PD, G-%PD, CCE,
and and
All other enzyme activities were measured with the methods of Colowick and Kaplan.8 Spectrophotometric methods were used throughout. Protein determinations were carried out with the biuret method. Enzymatic activity was initially developed as nanomoles of substrate consumed per milligram of protein per minute; in this presentation the activities are expressed as the per cent of controls. Statistical evaluation was performed with Student’s t test; results were considered significant when the p value was 0.05 or less. associates.’
Results Fig. 1 shows the comparative effects of progesterone on some enzymes related to lipogenesis. It will be seen that PFK, ME, G-6-PD, and CCE were elevated significantly after progesterone administration, while FAS and G-3-PD were unchanged. Fig. 2 shows the effects of progesterone on some of the enzymes related to gluconeogenesis. It can be seen that progesterone treatment results in increased PEPCK only. Comment The enzymatic patterns seen in these studies suggest that under the experimental conditions followed, progesterone administration causes increased activity of hepatic enzymes supporting the production of lipid precursors. PFK has long been recognized as being important in glycolysis.s It is of interest that insulin administration stimulates glycolysisi” and also stimulates PFK activity. ii As is seen from the data presented, as well as from the ensuing discussion, a role for insulin should be considered in the production of the observed changes. Lipid
synthesis
requires
reducing
equivalents,
pri-
Fig. 2. Effect zymes: LDH,
of progesterone on some PC, PEPCK, and FDPase.
“gluconeogenic”
en-
marily as reduced nicotinamide adenine dinucleotide phosphate (NADPH). The production of this substance is in part accomplished through the activity of ME (promoting the conversion of malate to pyruvate with NADPH as one of the reaction products) and, in part, through the activity of the pentose shunt dehydrogenases. Progesterone administration produces a significant increase in activity of ME and one of the pentose shunt dehydrogenases, G-6-PD. We have not studied 6-phosphogluconate dehydrogenase. The enzymatic pattern suggests increased NADPH production, thereby providing necessary precursors for lipid synthesis. Both MEi and G-6-PD levels are positively correlated with levels of insulin, though the increase of G-6-PD following insulin administration can be attributed to increased appetite.13 Nonetheless, there is a correlation between insulin levels and activity of these enzymes. CCE activity is also elevated following progesterone treatment. Since this enzyme promotes the formation of acetyl coenzyme A and oxalacetate from citrate, the enzymatic change renders it likely that additional lipid precursors (as acetyl coenzyme A) are being produced. CCE is also insulin responsive as shown by immunoassay and pulse-labeling studies.12 The enzymes discussed up to this point provide precursors for either fatty acids or cholesterol. There is no evidence, however, that fatty acid synthesis is promoted since the activity of FAS, which is directly involved in synthesis of fatty acids, is unchanged. The changes in enzymatic activity are similar to those reported as the result of insulin treatment, with the exception that FAS is unchanged. It has been reported previously that activity of this enzyme increases with insulin treatment but that the change is inhibited by simultaneous administration of glucagon.14 Whether the enzymatic pattern produced by progesterone treat-
132
Dahm,
ment mains
Minagawa,
is the result speculative;
to produce G-3-PD thesis
and Jellinek
of insulin-glucagon progesteronr
hyperinsulinemia.‘“, I6 has long been associated with
because
recently,
it
it promotes
glycerol
formation.”
shown
to
been from
glycerol
Our
kinase.”
of progesterone
generally
glycerol studies
treatment
Of the “gluconeogenic” one treatment resulted PC, and
glyceride
has
gluconeogenesis
L.DH,
interaction reis known. however,
FDP&e
considered
in
conjunction sh ow no significant
on this enzymes in elevation remained
to be a key
in
served
under the
tie” terone
enzyme
REFERENCES
1. Matute, M. I.., and Kalkhoff. R. K.: Sex ate]-oid influence on hepatic gluconeogenesis and glycogen f’ormation. Endocrinology 92: 762, 1973. 2. Sladek, C. D.: The effects of human chorionic somatomammotropin and estradiol on gluconeogenesis and hepatic glycogen formation in the rat, Hot-m. Mctab. Res. 7: 50, 1975. 3. Ballard. F. J., and Hanson, R. W.: The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxalacetate, J. Lipid Res. 8: 73. 1967. 4. Chang, H-C. and Lane, M.D.: The enzymatic carboxylation of phosphoenolpyruvate. Il. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase, J. Biol. Chem. 241: 2413. 1966. 5. Carlson. C. W., Tejwani, G. A.. Baxter, R. (:., Ulm, E. H.. and Pogell. B. W.: Involvement of cytosol proteins in oleate activation of rabbit liver fructose-l. 6-diphosphatase, J. Biol. Chem. 250: 4996, 1975. 6. Inoue. H.. Suzuki, F.. Fukunishi, K.. Adachi. K., and ‘l‘akeda. Y. Studies on ATP citrate lyasc of rat liver. I. Pwification and some properties, J.’ Biochem. (Tokyo) 60: 543. 1966. 7. Hsu, R. Y.. Wasson. G., and Porter. J. W.: The puriflcation and properties of the fatty acid synthetase of pigeon liver, J. Biol. Chem. 240: 3736, 1965. 8. Coltrwick, S.. and Kaplan, N.: Methoda in Enzymology, Nclc York, 1955, vol. 1. Academic Press, Inc. 9. (:ori, C. F.: Phosphorylation of carbohydrates. in A Synposium on Respiratory Enzymes. Madison, 1942. University of Wisconsin Press, p. 175. 10. Beitner, R., and Kalant. N.: Stimulation of glycolysis by insulin, J. Biol. Chem. 246: 500, 1971. 11. Weber, C.. Singhal. R. L.. Stamm, N. B.. Lea. M. A,, and Fisc-her. E. H.: Synchronous behavior pattern of key glycolytic enzymes: Glucokinase, phosphofructokinase and pyruvate kinase. Adv. Enzyme Regal. 4: 59. 1966.
this.
to the
capacity
hence,
with
which
fact,
has
formation
in PC: been
ob-
of
glucose-ti-phos-
be considered
of the
“dicarboxylic
capability
for
reported and
capability demonstrated
“”
pathways
may
by Matute been
changes always
glyconeogrnic
has been
gluconeogenic in
not
glucon~ogenrsis.‘“~
and
observations
treatment
hepatic is
up
while
have
favoring
gluconeogenic
increased and,
shuttle,”
activity
conditions
these
formation
PEPCK
acid
FDPase
identical
phate, lvith
with effect
studied. progestcrof PEPCK, while rate-limiting
and
are
More
enzyme.
unchanged.
“dicarboxylic
activity Since
syn-
participate
in the
consonant acid
increasrd following Kalkhoff.’
is also
shut-
glycogc-n progt’sInc-rrascd
suggested, in
rats
and tIllring
pregnancy.”
12. Gibson. D. M., Lyons. R. ‘1.. Scott, D. F., alltl Muto, Y.: Synthesis and degradation of the lipogenic enlyme\ 01 rat liver, Adv. Enzyme Regul. 10: 187, 1972. 13. Rudack, D.. Chisholm, E. M.. and Holten. D.: Rdt livrr glucose 6-phosphate dehydrogenase. Regulation bv carbohydrate diet and insulin, J. Biol. Chem. 246: 1249. 1971. 14. I.akshmanan, M. R.. Nepokroeff, C. M., and I’or-trr, J. W.: Control of the synthesis of fatty-acid synthetasc in rat liver by insulin, glucagon and adenosine 3’:j’ cyclic monophosphate, Proc. Natl. Acad. Sci. 69: 3.~16, 1972. 15. Beck, P.: Progestin enhancement of the plasma insulin rcsponsc to glucose in rhesus monkeys, Diabetes 18: 136. 1969. 6. Costrini, N. V., and Kalkhoff. R. K.: Rclatii-c ef frc ts of pregnancy, estradiol, and progesterone on plasma insulin and pancreatic islet secretion, J. Clin. lnve\t. 50: 992. 1971. 7. Weiss, S. B.. and Kennedy, I-. F.: The cn7ymatic svnthcsiy of triglycerides, J, Am. Chem. Sot. 78: 3550. 1956. 18. Harding, J. W., Jr,, Pyeritz. E. A.. Morris, H. P., and White, H. B.. III: Proportional activities of glvccrol kinasr and glycerol 3-phosphate dehydrogenase in rar hepatomas, Biochem. J. 148: 545, 1975. 19. Perrt. J., and Chanez. M.: InHurnce of diet, cot-t& and insulin on the activity of pyruvate carboxvlasc and phosphoenolpyruvate carbox&inase in the ra; li\ct-. J. %uct-. 106: 103. 1976. 20. Exton, J. H., Mallette, I.. E.. Jefferson. 1.. S.. U’ong. k.. H. A.. Friedmann. N.. Miller, T. B...Jr.. and Park, C. R.: ‘The hormonal control of hepatic gluconeogenrair. Recent PI-og. Horm. Res. 26: 411, 1970. 21. Herrera, E., Knopp, R. H.. and Freinkrl. N.: (Zarbohcdrate metabolism in pregnancy. VI. Plasma fuels, insulin. liver composition, gluconeogenesis and nitrogen mrtabolism during late gestation in the fed and fasted rat. J. Clin. Invest. 48: 2260. 1969.
H.
JUNNOSUKE MAX
DAHM,
JR.,
M.D.
MINAGAWA,
JELLINEK,
M.D.
PH.D.
St. Louis. Missouri The known effect of progesterone on carbohydrate metabolism prompted a study of some of the hepatic “lipogenic” and “gluconeogenic” enzymes in rats treated with progesterone. Several enzymes providing lipid precursors (phosphofructokinase, malic enzyme, glucose-6-phosphate dehydrogenase, and citrate cleavage enzyme) showed increased specific activity. These changes may represent insulin effects. Specific activity of phosphoenolpyruvate carboxykinase, usually associated with control of gluconeogenesis, was also increased. The latter is compatible with increased capability for glycogenesis, which is recognized as a progesterone effect. (AM. J. OBSTET. GYNECOL. 129: 130, 1977.)
taneously. for 14 days, followed by 2.5 mg. twice daily for seven days. The animals were killed after 2 1 days of treatment. Sodium pentobarbital was given intraperitoneally in a dose of 40 mg. per kilogram. After anesthesia was induced, the livers were excised, blotted free of blood, and placed on ice. A 10 per cent w/v liver homogenate was prepared in cold 30 per cent sucrose solution in a Dual1 homogenizer. The soluble fraction was then separated by ultracentrifugation at 40,000 r.p.m. for one hour. The resultant pellet was resuspended in 12 ml. of cold sucrose solution, and cellular debris was removed by centrifugation at 8,000 r.p.m. for 10 minutes. The supernatant containing the mitochondria was then recentrifuged at 25,000 r.p.m. for 30 minutes. The mitochondrial pellet was resuspended in 5 ml. of 30 per cent sucrose solution and the mitochondrial membrane was disrupted by sonication for approximately one minute. Enzymes studied included phosphofructokinase (PFK), malic enzyme (ME), glucose-6-phosphate dehydrogenase (G-6-PD), glycerol-3-phosphate dehydrogenase (G-3-PD), citrate cleavage enzyme (CCE), fatty acid synthetase (FAS). pyruvate carboxylase (PC). phosphoenolpyruvate carboxykinase (PEPCK), fructose- 1,6-diphosphatase (FDPase), and lactate dehydrogenase (LDH). PC was measured by the method of Ballard and Hanson3 PEPCK was measured by the method of Chang and Lane,4 and FDPase was measured by the method of Carlson and associates.5 CCE was measured by the method of Inoue and associateQ while FAS was determined by the method of Hsu and
that progeSterOne influences the intermediary metabolism of carbohydrate in the liver. Matute and Kalkhoff’ have shown that progesterone, given singly or in combination with estradiol, suppresses the incorporation of alanineU”‘C and pyruvate-3-i% into glucose. However, hepatic glycogen deposition is increased. Sladek’ has shown that, while gluconeogenesis from alanine is inhibited by estradiol administration, incorporation of alanine into glycogen is increased. The present investigation was undertaken to study the effects of progesterone on some of the hepatic enzymes associated with lipogenesis and gluconeogenesis in rats. IT
IS
GENERALLY
ACCEPTED
Methods Intact female Wistar rats weighing approximately 200 grams were used. Twenty-four animals were studied in all determinations. Food and water were provided freely throughout the course of the treatment. Progesterone (12.5 mg. per milliliter of sesame oil) was given in a dose of 1.25 mg. twice daily, subcuFrom the Departments of Obstetrics-Gynecology Surgery, St. Louis University, and St. Louis Hospital. Supported in part No. HLO-6312-15. Received
for
publication
Accepted April Reprint
by National March
Institutes
and City
of Health
Grant
4, 1977.
14. 1977.
requests: Dr. Charles
of Obstetrics
and
Louis University, Missouri 63104.
H. Dahm, Jr., Department School of Medicine, St. 132.5 S. Grand Blvd., St. Louis,
Gynecology,
130
Volume Number
129 2
Progesterone effects on metabolism
Control
cl
Progesterone
m
p
‘P <0.005 “P io.COo5
2
.cl
q
Control
131
‘P
Progesterone
PC
Fig. 1. Effect of progesterone “lipogenic” enzymes: PFK, ME, FAS.
on some “glycolytic” G-6-PD, G-%PD, CCE,
and and
All other enzyme activities were measured with the methods of Colowick and Kaplan.8 Spectrophotometric methods were used throughout. Protein determinations were carried out with the biuret method. Enzymatic activity was initially developed as nanomoles of substrate consumed per milligram of protein per minute; in this presentation the activities are expressed as the per cent of controls. Statistical evaluation was performed with Student’s t test; results were considered significant when the p value was 0.05 or less. associates.’
Results Fig. 1 shows the comparative effects of progesterone on some enzymes related to lipogenesis. It will be seen that PFK, ME, G-6-PD, and CCE were elevated significantly after progesterone administration, while FAS and G-3-PD were unchanged. Fig. 2 shows the effects of progesterone on some of the enzymes related to gluconeogenesis. It can be seen that progesterone treatment results in increased PEPCK only. Comment The enzymatic patterns seen in these studies suggest that under the experimental conditions followed, progesterone administration causes increased activity of hepatic enzymes supporting the production of lipid precursors. PFK has long been recognized as being important in glycolysis.s It is of interest that insulin administration stimulates glycolysisi” and also stimulates PFK activity. ii As is seen from the data presented, as well as from the ensuing discussion, a role for insulin should be considered in the production of the observed changes. Lipid
synthesis
requires
reducing
equivalents,
pri-
Fig. 2. Effect zymes: LDH,
of progesterone on some PC, PEPCK, and FDPase.
“gluconeogenic”
en-
marily as reduced nicotinamide adenine dinucleotide phosphate (NADPH). The production of this substance is in part accomplished through the activity of ME (promoting the conversion of malate to pyruvate with NADPH as one of the reaction products) and, in part, through the activity of the pentose shunt dehydrogenases. Progesterone administration produces a significant increase in activity of ME and one of the pentose shunt dehydrogenases, G-6-PD. We have not studied 6-phosphogluconate dehydrogenase. The enzymatic pattern suggests increased NADPH production, thereby providing necessary precursors for lipid synthesis. Both MEi and G-6-PD levels are positively correlated with levels of insulin, though the increase of G-6-PD following insulin administration can be attributed to increased appetite.13 Nonetheless, there is a correlation between insulin levels and activity of these enzymes. CCE activity is also elevated following progesterone treatment. Since this enzyme promotes the formation of acetyl coenzyme A and oxalacetate from citrate, the enzymatic change renders it likely that additional lipid precursors (as acetyl coenzyme A) are being produced. CCE is also insulin responsive as shown by immunoassay and pulse-labeling studies.12 The enzymes discussed up to this point provide precursors for either fatty acids or cholesterol. There is no evidence, however, that fatty acid synthesis is promoted since the activity of FAS, which is directly involved in synthesis of fatty acids, is unchanged. The changes in enzymatic activity are similar to those reported as the result of insulin treatment, with the exception that FAS is unchanged. It has been reported previously that activity of this enzyme increases with insulin treatment but that the change is inhibited by simultaneous administration of glucagon.14 Whether the enzymatic pattern produced by progesterone treat-
132
Dahm,
ment mains
Minagawa,
is the result speculative;
to produce G-3-PD thesis
and Jellinek
of insulin-glucagon progesteronr
hyperinsulinemia.‘“, I6 has long been associated with
because
recently,
it
it promotes
glycerol
formation.”
shown
to
been from
glycerol
Our
kinase.”
of progesterone
generally
glycerol studies
treatment
Of the “gluconeogenic” one treatment resulted PC, and
glyceride
has
gluconeogenesis
L.DH,
interaction reis known. however,
FDP&e
considered
in
conjunction sh ow no significant
on this enzymes in elevation remained
to be a key
in
served
under the
tie” terone
enzyme
REFERENCES
1. Matute, M. I.., and Kalkhoff. R. K.: Sex ate]-oid influence on hepatic gluconeogenesis and glycogen f’ormation. Endocrinology 92: 762, 1973. 2. Sladek, C. D.: The effects of human chorionic somatomammotropin and estradiol on gluconeogenesis and hepatic glycogen formation in the rat, Hot-m. Mctab. Res. 7: 50, 1975. 3. Ballard. F. J., and Hanson, R. W.: The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxalacetate, J. Lipid Res. 8: 73. 1967. 4. Chang, H-C. and Lane, M.D.: The enzymatic carboxylation of phosphoenolpyruvate. Il. Purification and properties of liver mitochondrial phosphoenolpyruvate carboxykinase, J. Biol. Chem. 241: 2413. 1966. 5. Carlson. C. W., Tejwani, G. A.. Baxter, R. (:., Ulm, E. H.. and Pogell. B. W.: Involvement of cytosol proteins in oleate activation of rabbit liver fructose-l. 6-diphosphatase, J. Biol. Chem. 250: 4996, 1975. 6. Inoue. H.. Suzuki, F.. Fukunishi, K.. Adachi. K., and ‘l‘akeda. Y. Studies on ATP citrate lyasc of rat liver. I. Pwification and some properties, J.’ Biochem. (Tokyo) 60: 543. 1966. 7. Hsu, R. Y.. Wasson. G., and Porter. J. W.: The puriflcation and properties of the fatty acid synthetase of pigeon liver, J. Biol. Chem. 240: 3736, 1965. 8. Coltrwick, S.. and Kaplan, N.: Methoda in Enzymology, Nclc York, 1955, vol. 1. Academic Press, Inc. 9. (:ori, C. F.: Phosphorylation of carbohydrates. in A Synposium on Respiratory Enzymes. Madison, 1942. University of Wisconsin Press, p. 175. 10. Beitner, R., and Kalant. N.: Stimulation of glycolysis by insulin, J. Biol. Chem. 246: 500, 1971. 11. Weber, C.. Singhal. R. L.. Stamm, N. B.. Lea. M. A,, and Fisc-her. E. H.: Synchronous behavior pattern of key glycolytic enzymes: Glucokinase, phosphofructokinase and pyruvate kinase. Adv. Enzyme Regal. 4: 59. 1966.
this.
to the
capacity
hence,
with
which
fact,
has
formation
in PC: been
ob-
of
glucose-ti-phos-
be considered
of the
“dicarboxylic
capability
for
reported and
capability demonstrated
“”
pathways
may
by Matute been
changes always
glyconeogrnic
has been
gluconeogenic in
not
glucon~ogenrsis.‘“~
and
observations
treatment
hepatic is
up
while
have
favoring
gluconeogenic
increased and,
shuttle,”
activity
conditions
these
formation
PEPCK
acid
FDPase
identical
phate, lvith
with effect
studied. progestcrof PEPCK, while rate-limiting
and
are
More
enzyme.
unchanged.
“dicarboxylic
activity Since
syn-
participate
in the
consonant acid
increasrd following Kalkhoff.’
is also
shut-
glycogc-n progt’sInc-rrascd
suggested, in
rats
and tIllring
pregnancy.”
12. Gibson. D. M., Lyons. R. ‘1.. Scott, D. F., alltl Muto, Y.: Synthesis and degradation of the lipogenic enlyme\ 01 rat liver, Adv. Enzyme Regul. 10: 187, 1972. 13. Rudack, D.. Chisholm, E. M.. and Holten. D.: Rdt livrr glucose 6-phosphate dehydrogenase. Regulation bv carbohydrate diet and insulin, J. Biol. Chem. 246: 1249. 1971. 14. I.akshmanan, M. R.. Nepokroeff, C. M., and I’or-trr, J. W.: Control of the synthesis of fatty-acid synthetasc in rat liver by insulin, glucagon and adenosine 3’:j’ cyclic monophosphate, Proc. Natl. Acad. Sci. 69: 3.~16, 1972. 15. Beck, P.: Progestin enhancement of the plasma insulin rcsponsc to glucose in rhesus monkeys, Diabetes 18: 136. 1969. 6. Costrini, N. V., and Kalkhoff. R. K.: Rclatii-c ef frc ts of pregnancy, estradiol, and progesterone on plasma insulin and pancreatic islet secretion, J. Clin. lnve\t. 50: 992. 1971. 7. Weiss, S. B.. and Kennedy, I-. F.: The cn7ymatic svnthcsiy of triglycerides, J, Am. Chem. Sot. 78: 3550. 1956. 18. Harding, J. W., Jr,, Pyeritz. E. A.. Morris, H. P., and White, H. B.. III: Proportional activities of glvccrol kinasr and glycerol 3-phosphate dehydrogenase in rar hepatomas, Biochem. J. 148: 545, 1975. 19. Perrt. J., and Chanez. M.: InHurnce of diet, cot-t& and insulin on the activity of pyruvate carboxvlasc and phosphoenolpyruvate carbox&inase in the ra; li\ct-. J. %uct-. 106: 103. 1976. 20. Exton, J. H., Mallette, I.. E.. Jefferson. 1.. S.. U’ong. k.. H. A.. Friedmann. N.. Miller, T. B...Jr.. and Park, C. R.: ‘The hormonal control of hepatic gluconeogenrair. Recent PI-og. Horm. Res. 26: 411, 1970. 21. Herrera, E., Knopp, R. H.. and Freinkrl. N.: (Zarbohcdrate metabolism in pregnancy. VI. Plasma fuels, insulin. liver composition, gluconeogenesis and nitrogen mrtabolism during late gestation in the fed and fasted rat. J. Clin. Invest. 48: 2260. 1969.