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
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
