OVINE PLACENTAL LACTOGEN: ACUTE EFFECTS ON INTERMEDIARY METABOLISM IN PREGNANT AND NON-PREGNANT SHEEP
STUART HANDWERGER, R. E. FELLOWS, M. C. CRENSHAW, THOMAS HURLEY, JANET BARRETT AND W. F. MAURER The Departments of Pediatrics, Physiology, Obstetrics and Gynecology, and Medicine, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
(Received 14 July 1975) SUMMARY
The intravenous administration of ovine placental lactogen to pregnant and non-pregnant sheep produced significant acute decreases in plasma free fatty acid, glucose and amino nitrogen concentrations. Plasma insulin concentrations decreased 1 h after administration of ovine placental lactogen and then increased significantly above baseline concentrations. The results suggest that, like human placental lactogen, ovine placental lactogen is important in the modulation of intermediary metabolism during pregnancy. The sheep is an excellent animal model for the investigation of the physiology of placental lactogen.
INTRODUCTION
In previous reports from our laboratories, ovine placental lactogen, isolated and purified from sheep cotyledons, was shown to have chemical and biological properties resembling human placental lactogen (Handwerger, Maurer, Barrett, Hurley & Fellows, 1974; Hurley, Fellows, Maurer & Handwerger, 1975). Like human placental lactogen, ovine placental lactogen will compete for membrane binding sites with human prolactin and human growth hormone and will stimulate lactation in vivo and in vitro (Handwerger et al. 1974). Numerous studies indicate that, in addition to its effects on lactation, placental lactogen is important in the modulation of maternal carbohydrate, lipid and protein metabolism during human pregnancy (Grumbach, Kaplan, Sciarra & Burr, 1968). This communication reports the first studies of the acute effects of ovine placental lactogen on intermediary metabolism in the sheep. Partially purified ovine placental lactogen was
administered intravenously on multiple occasions to both pregnant and non-pregnant sheep and plasma free fatty acid, insulin, glucose and amino nitrogen concentrations were deter¬ mined before and after administration of the hormone. ANIMALS AND METHODS
Three non-pregnant and two pregnant Dorset ewes weighing 41 to 61 kg-were studied. One pregnant ewe was investigated from days 85 to 135 of pregnancy and delivered a full-term normal male lamb on day 143. The second pregnant ewe, studied from days 80 to 100 of * Presented in part at the Third International Symposium on Growth Hormone and Related Peptides, Milan, Italy, 24 September 1975. t Reprint requests should be addressed to Dr Handwerger, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
pregnancy, delivered prematurely on day 124. All sheep were housed in individual stalls throughout the experimental period and were fed a diet of hay and mixed grains with vitamin and mineral supplements. Indwelling polyvinyl chloride catheters were placed in a femoral vein of each animal at least 1 week before study. The ovine placental lactogen was isolated and partially purified from sheep cotyledons by ammonium bicarbonate extraction, ammonium sulphate fractionation, and chromatography on Sephadex G-150 as previously described (Handwerger et al. 1974; Hurley et al. 1975). The active material was estimated to be up to 60 % pure in the mammary gland prolactin receptor assay using purified human placental lactogen as standard. The material showed no cross-reactivity by radial immunodiffusion with antisera raised against either ovine growth hormone or ovine prolactin and was free of insulin, ovine growth hormone and ovine prolactin by radio¬
immunoassay. After an overnight fast, the sheep were placed in portable cages large enough to permit standing and reclining. Thirty to 45 min later, ovine placental lactogen dissolved in 10 ml phosphate-buffered saline or 10 ml buffered saline alone was administered over 3-5 min through the indwelling femoral catheter. The ovine placental lactogen was dissolved and sterilized by Millipore filtration immediately before administration. Blood samples were collected immediately before and 1, 2, 3, 4, 6 and 8 h after injection and the plasma was frozen at
70 °C until assayed. The minimum interval between repeated studies in the 4 days. sheep Glucose was determined by the glucose oxidase method using a Beckman Glucose Analyzer, free fatty acids by the method of Dole (1956), amino nitrogen by the method of Goodwin (1968), and insulin by radioimmunoassay (Morgan & Lazarow, 1963). The statistical difference between sample means was determined by Student's /-test. —
same
was
RESULTS
The two pregnant sheep received 50 mg ovine placental lactogen in five experiments and buffered saline as control in four experiments, while the three non-pregnant sheep received 50 mg ovine placental lactogen and saline each in four experiments. No significant difference was noted between the response of the non-pregnant and pregnant sheep. In all experiments, the intravenous administration of 50 mg ovine placental lactogen (0-82-1-22 mg/kg body weight) caused significant changes in the plasma concentrations of free fatty acids, insulin, glucose and amino nitrogen (Fig. 1). Free fatty acid concentrations decreased 64-4 ±4-5 (s.e.m.) % 1 h after injection of ovine placental lactogen and did not return to the baseline concentrations until 6 h later. Insulin concentrations fell 67-7 ±6-7% at lh and then increased progressively, reaching 150-0 ±26-4% above baseline concentrations at 8 h. In contrast, insulin levels in the control experiments decreased during the 8 h interval. Plasma glucose and amino nitrogen concentrations remained unchanged for the first 2 h after administration of ovine placental lactogen and then declined gradually over the next 6 h. Eight hours after the administration of ovine placental lactogen, plasma glucose concentra¬ tions had decreased 40-8 ±3-7% and plasma amino nitrogen levels had decreased 41-0 ±2-7%. To establish a dose-response relationship, ovine placental lactogen was also administered to each of the two pregnant ewes in single doses of 1,5,10,25 and 50 mg. A statistically significant negative correlation was noted between the logarithm of the dose of ovine placental lactogen and the magnitude of the change in free fatty acid, glucose and amino nitrogen concentrations from baseline concentrations (Fig. 2). Compared with the saline control, 1 mg ovine placental lactogen caused no significant changes in the plasma concentrations of free fatty acids, insulin, glucose or amino nitrogen. Five milligrams ovine placental lactogen caused modest
but statistically significant decreases in the concentrations of free fatty acids and amino nitrogen without significant effects on plasma glucose and insulin concentrations, while 10 to 50 mg ovine placental lactogen produced changes in the concentrations of all four.
Fig. 1. Effect of ovine placental lactogen on intermediary metabolism in the sheep. (A) Free fatty acids and insulin; (B) glucose and amino nitrogen. At time 0, either 50 mg ovine placental lactogen (9 experiments) or buffered saline (8 experiments) was administered intravenously to two pregnant and three non-pregnant sheep over a 2-3 min interval. No significant differences were noted between the responses of the pregnant and non-pregnant sheep. The shaded area represents the mean ± s.e.m.
of the response to buffered saline. The bars enclose the mean ± s.e.m. of the response to ovine placental lactogen. Baseline concentrations (means ± s.e.m.) in the non-pregnant sheep: free fatty acids 1169+186/¿equiv./I; insulin 15-0±2-2 /iu./ml; glucose 59-4+1-6 mg/di; amino nitrogen 4-27 + 016 mg/dl. Baseline concentrations in the pregnant sheep : free fatty acids 1055 ±111 /¿equiv./l ; insulin 7-7 + 1-1 /tu./ml; glucose 63-3± 1-7 mg/dl; amino nitrogen 3-89 + 0-09 mg/dl. *P < 005; **P < 0005; ***P < 0001. Free
(B)
fatty acid
Glucose r=0-94
F=0-02
-20
25
50
0
1
10
25
50
Ovine placental lactogen (mg) Fig. 2. Relationship between log of the dose'of ovine placental lactogen and the maximum response, expressed as percent of change from baseline concentration. (A) Free fatty acids and insulin; (B) glucose and amino nitrogen. Slopes were calculated by linear regression analysis.
DISCUSSION
Although placental lactogens have been identified in man (Josimovich & McLaren, 1962), monkeys (Shome & Friesen, 1971), and two other subprimate groups, the rodents (Ray, Averill, Lyons & Johnson, 1955; Kelly, Shiu, Robertson & Friesen, 1975) and domestic ruminants (Buttle, Forsyth & Knaggs, 1972; Kelly, Robertson & Friesen, 1974), only the metabolic effects of human placental lactogen have been studied previously. The administra¬ tion of human placental lactogen to human subjects has been demonstrated to cause deterioration in glucose tolerance and an increase in the insulin response to glucose (Beck & Daughaday, 1967; Grumbach et al. 1968). In experiments in vitro, human placental lactogen has been shown to enhance insulin secretion from isolated pancreatic slices (Martin & Friesen, 1969). The effects of human placental lactogen on lipid metabolism are less clear. Prolonged intramuscular administration causes an increase in free fatty acid concentrations (Grumbach, Kaplan, Abrams, Beel & Conte, 1966) but intravenous administration of 4 mg (Berle, Finsterwalder & Apostolakis, 1974) has no effect on free fatty acid concentrations. In vitro, human placental lactogen stimulates both lipolysis (Turtle & Kipnis, 1967; Genazzani, Benuzzi-Badoni & Felber, 1969) and lipogenesis (Felber, Zaragoza, Benuzzi-Badoni & Genazzani, 1972). Our results indicate that ovine, like human placental lactogen, has significant effects on intermediary metabolism although the mechanisms by which these effects are exerted are obscure. After administration of ovine placental lactogen there were acute decreases in both free fatty acid and insulin concentrations. Thereafter, free fatty acid concentrations returned to baseline even though there was a progressive increase in insulin secretion. Plasma glucose and amino nitrogen concentrations began to decline about 1 h after the rise in insulin secretion. Although insulin administration has been demonstrated to cause a decrease in amino nitrogen (Luck, Morrison & Wilbur, 1928), the decrease in amino nitrogen following administration of ovine placental lactogen cannot be attributed entirely to insulin since 5 mg ovine placental lactogen caused a decrease in amino nitrogen concentrations without a change in insulin concentrations. The prolonged effects of ovine placental lactogen administration on insulin, glucose, and amino nitrogen metabolism are striking. Although we have no explanation as yet for these prolonged effects, it is of interest that acute administration of ovine growth hormone and prolactin has also been reported to produce prolonged metabolic effects lasting as long as 24 h (Manns & Boda, 1965; Winkler, Rothgeb, Steele & Altszuler, 1971). It is also of interest that acute administration of ovine placental lactogen caused nearly identical effects in the pregnant and non-pregnant sheep. Studies on chronic administration of ovine placental lactogen in pregnant and non-pregnant sheep are in progress. Grumbach et al. (1968) have postulated that human placental lactogen, by its anabolic and diabetogenic effects, acts metabolically as the growth hormone of pregnancy and permits the foetus to receive an adequate supply of glucose and amino acids. The striking effects of a preparation of ovine placental lactogen free of growth hormone, prolactin, and insulin on carbohydrate, lipid and amino acid metabolism in the pregnant sheep also provide strong support for the thesis that ovine placental lactogen is important in the regulation of intermediary metabolism during pregnancy. The sheep, therefore, provides an excellent animal model for investigation of the physiological role of placental lactogen in maternal and foetal metabolism and the mechanisms by which placental lactogen exerts its metabolic effects.
We thank James B. Sidbury, Harold Lebovitz and George Brumley for helpful discussions, and Elaine Haynes, Rebecca Bowen and Larry Kodack for technical assistance. This work was supported by grants from the National Institutes of Health (HD 07447 and AM 12861) and the National Foundation-March of Dimes (CRBS-297). S.H. is the recipient of a Career Development Award from the U.S. Public Health Service (HD 00065). REFERENCES
Daughaday, W. H. (1967). Human placental lactogen: studies of its acute metabolic effects and Beck, disposition in normal man. Endocrinology 46,103-109. Berle, P., Finsterwalder, E. & Apostolakis, M. (1974). Comparative studies on the effect of human growth hormone, human prolactin and human placental lactogen on lipid metabolism. Hormone and Metabolic Research 6, 347-350. Buttle, H. L., Forsyth, I. A. & Knaggs, G. S. (1972). Plasma prolactin measured by radioimmunoassay and bioassay in pregnant and lactating goats and the occurrence of a placental lactogen. Journal of Endocrinology 53, 483-491. Dole, V. P. (1956). A relation between non-esterified fatty acids in plasma and the metabolism of glucose. Journal of Clinical Investigation 35, 150-154. Felber, J. P., Zaragoza, ., Benuzzi-Badoni, M. & Genazzani, A. R. (1972). The double effect of human chorionic somatomammotropin (HCS) and pregnancy on lipogenesis and lipolysis in the isolated rat epididymal fat pad and fat pad cells. Hormone and Metabolic Research 4, 293-296. Genazzani, A. R., Benuzzi-Badoni, M. & Felber, J. P. (1969). Human chorionic somato-mammotropin (HCSM): lipolytic action of a pure preparation on isolated fat cells. Metabolism 18, 593-598. Goodwin, J. F. (1968). The colorimetrie estimation of plasma amino nitrogen with DNFB. Clinical Chemistry 14, 1080-1090. Grumbach, M. M., Kaplan, S. L., Abrams, C. L., Beel, J. J. & Conte, F. . (1966). Plasma free fatty acid response to the administration of chorionic growth hormone-prolactin. Journal of Clinical Endocrinology and Metabolism 26, 478^182. Grumbach, M. M., Kaplan, S. L., Sciarra, J. J. & Burr, I. M. (1968). Chorionic growth hormone prolactin (CGP): secretion, disposition, biologic activity in man, and postulated function as the 'growth hormone' of the second half of pregnancy. Annals of New York Academy of Sciences 148, 501-531. Handwerger, S., Maurer, W., Barrett, J., Hurley, T. & Fellows, R. E. (1974). Evidence for homology between ovine and human placental lactogens. Endocrine Research Communications 1, 401-413. Hurley, T., Fellows, R. E., Maurer, W. F. & Handwerger, S. (1975). Ovine placental lactogen: structural and functional relationship with primate placental lactogen, growth hormone and prolactin. In Fourth American Peptide Symposium, ed. R. Walter. Ann Arbor: Ann Arbor Science Publishers (in Press). Josimovich, J. B. & McLaren, J. A. (1962). Presence in the human placenta and term serum of a highly lactogenic substance immunologically related to pituitary growth hormone. Endocrinology 71, 209-220. Kelly, P. ., Robertson, H. A. & Friesen, H. G. (1974). Temporal pattern of placental lactogen and pro¬ gesterone secretion in sheep. Nature 248, 435-437. Kelly, P. ., Shiu, R. P. C, Robertson, H. A. & Friesen, H. G. (1975). Characterization of rat chorionic mammotropin. Endocrinology 96, 1187-1195. Luck, J. M., Morrison, G. & Wilbur, L. F. (1928). Effect of insulin on the amino acid content of blood. Journal of Biological Chemistry 77,151-156. Manns, J. G. & Boda, J. M. (1965). Effects of ovine growth hormone and prolactin on blood glucose, serum insulin, plasma nonesterified fatty acids and amino nitrogen in sheep. Endocrinology 76, 1109-1114. Martin, J. M. & Friesen, H. G. (1969). Effect of human placental lactogen on the isolated islets of Langerhans in vitro. Endocrinology 84, 619-621. Morgan, C. R. & Lazarow, A. (1963). Immunoassay of insulin: two antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12, 115-126. Ray, E. W., Averill, S. C, Lyons, W. R. & Johnson, R. E. (1955). Rat placental hormonal activities corre¬ sponding to those of pituitary mammotropin. Endocrinology 56, 359-373. Shome, . & Friesen, H. G. (1971). Purification and characterization of monkey placental lactogen. Endocrinology 89, 631-641. Turtle, J. R. & Kipnis, D. M. (1967). The lipolytic action of human placental lactogen on isolated fat cells. Biochimica et Biophysica Acta 144, 583-594. Winkler, B., Rothgeb, L, Steele, R. & Altszuler, N. (1971). Effect of ovine prolactin administration on free fatty acid metabolism in the normal dog. Endocrinology 88, 1349-1352. P. &
STUART HANDWERGER, R. E. FELLOWS, M. C. CRENSHAW, THOMAS HURLEY, JANET BARRETT AND W. F. MAURER The Departments of Pediatrics, Physiology, Obstetrics and Gynecology, and Medicine, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
(Received 14 July 1975) SUMMARY
The intravenous administration of ovine placental lactogen to pregnant and non-pregnant sheep produced significant acute decreases in plasma free fatty acid, glucose and amino nitrogen concentrations. Plasma insulin concentrations decreased 1 h after administration of ovine placental lactogen and then increased significantly above baseline concentrations. The results suggest that, like human placental lactogen, ovine placental lactogen is important in the modulation of intermediary metabolism during pregnancy. The sheep is an excellent animal model for the investigation of the physiology of placental lactogen.
INTRODUCTION
In previous reports from our laboratories, ovine placental lactogen, isolated and purified from sheep cotyledons, was shown to have chemical and biological properties resembling human placental lactogen (Handwerger, Maurer, Barrett, Hurley & Fellows, 1974; Hurley, Fellows, Maurer & Handwerger, 1975). Like human placental lactogen, ovine placental lactogen will compete for membrane binding sites with human prolactin and human growth hormone and will stimulate lactation in vivo and in vitro (Handwerger et al. 1974). Numerous studies indicate that, in addition to its effects on lactation, placental lactogen is important in the modulation of maternal carbohydrate, lipid and protein metabolism during human pregnancy (Grumbach, Kaplan, Sciarra & Burr, 1968). This communication reports the first studies of the acute effects of ovine placental lactogen on intermediary metabolism in the sheep. Partially purified ovine placental lactogen was
administered intravenously on multiple occasions to both pregnant and non-pregnant sheep and plasma free fatty acid, insulin, glucose and amino nitrogen concentrations were deter¬ mined before and after administration of the hormone. ANIMALS AND METHODS
Three non-pregnant and two pregnant Dorset ewes weighing 41 to 61 kg-were studied. One pregnant ewe was investigated from days 85 to 135 of pregnancy and delivered a full-term normal male lamb on day 143. The second pregnant ewe, studied from days 80 to 100 of * Presented in part at the Third International Symposium on Growth Hormone and Related Peptides, Milan, Italy, 24 September 1975. t Reprint requests should be addressed to Dr Handwerger, Department of Pediatrics, Duke University Medical Center, Durham, North Carolina 27710, U.S.A.
pregnancy, delivered prematurely on day 124. All sheep were housed in individual stalls throughout the experimental period and were fed a diet of hay and mixed grains with vitamin and mineral supplements. Indwelling polyvinyl chloride catheters were placed in a femoral vein of each animal at least 1 week before study. The ovine placental lactogen was isolated and partially purified from sheep cotyledons by ammonium bicarbonate extraction, ammonium sulphate fractionation, and chromatography on Sephadex G-150 as previously described (Handwerger et al. 1974; Hurley et al. 1975). The active material was estimated to be up to 60 % pure in the mammary gland prolactin receptor assay using purified human placental lactogen as standard. The material showed no cross-reactivity by radial immunodiffusion with antisera raised against either ovine growth hormone or ovine prolactin and was free of insulin, ovine growth hormone and ovine prolactin by radio¬
immunoassay. After an overnight fast, the sheep were placed in portable cages large enough to permit standing and reclining. Thirty to 45 min later, ovine placental lactogen dissolved in 10 ml phosphate-buffered saline or 10 ml buffered saline alone was administered over 3-5 min through the indwelling femoral catheter. The ovine placental lactogen was dissolved and sterilized by Millipore filtration immediately before administration. Blood samples were collected immediately before and 1, 2, 3, 4, 6 and 8 h after injection and the plasma was frozen at
70 °C until assayed. The minimum interval between repeated studies in the 4 days. sheep Glucose was determined by the glucose oxidase method using a Beckman Glucose Analyzer, free fatty acids by the method of Dole (1956), amino nitrogen by the method of Goodwin (1968), and insulin by radioimmunoassay (Morgan & Lazarow, 1963). The statistical difference between sample means was determined by Student's /-test. —
same
was
RESULTS
The two pregnant sheep received 50 mg ovine placental lactogen in five experiments and buffered saline as control in four experiments, while the three non-pregnant sheep received 50 mg ovine placental lactogen and saline each in four experiments. No significant difference was noted between the response of the non-pregnant and pregnant sheep. In all experiments, the intravenous administration of 50 mg ovine placental lactogen (0-82-1-22 mg/kg body weight) caused significant changes in the plasma concentrations of free fatty acids, insulin, glucose and amino nitrogen (Fig. 1). Free fatty acid concentrations decreased 64-4 ±4-5 (s.e.m.) % 1 h after injection of ovine placental lactogen and did not return to the baseline concentrations until 6 h later. Insulin concentrations fell 67-7 ±6-7% at lh and then increased progressively, reaching 150-0 ±26-4% above baseline concentrations at 8 h. In contrast, insulin levels in the control experiments decreased during the 8 h interval. Plasma glucose and amino nitrogen concentrations remained unchanged for the first 2 h after administration of ovine placental lactogen and then declined gradually over the next 6 h. Eight hours after the administration of ovine placental lactogen, plasma glucose concentra¬ tions had decreased 40-8 ±3-7% and plasma amino nitrogen levels had decreased 41-0 ±2-7%. To establish a dose-response relationship, ovine placental lactogen was also administered to each of the two pregnant ewes in single doses of 1,5,10,25 and 50 mg. A statistically significant negative correlation was noted between the logarithm of the dose of ovine placental lactogen and the magnitude of the change in free fatty acid, glucose and amino nitrogen concentrations from baseline concentrations (Fig. 2). Compared with the saline control, 1 mg ovine placental lactogen caused no significant changes in the plasma concentrations of free fatty acids, insulin, glucose or amino nitrogen. Five milligrams ovine placental lactogen caused modest
but statistically significant decreases in the concentrations of free fatty acids and amino nitrogen without significant effects on plasma glucose and insulin concentrations, while 10 to 50 mg ovine placental lactogen produced changes in the concentrations of all four.
Fig. 1. Effect of ovine placental lactogen on intermediary metabolism in the sheep. (A) Free fatty acids and insulin; (B) glucose and amino nitrogen. At time 0, either 50 mg ovine placental lactogen (9 experiments) or buffered saline (8 experiments) was administered intravenously to two pregnant and three non-pregnant sheep over a 2-3 min interval. No significant differences were noted between the responses of the pregnant and non-pregnant sheep. The shaded area represents the mean ± s.e.m.
of the response to buffered saline. The bars enclose the mean ± s.e.m. of the response to ovine placental lactogen. Baseline concentrations (means ± s.e.m.) in the non-pregnant sheep: free fatty acids 1169+186/¿equiv./I; insulin 15-0±2-2 /iu./ml; glucose 59-4+1-6 mg/di; amino nitrogen 4-27 + 016 mg/dl. Baseline concentrations in the pregnant sheep : free fatty acids 1055 ±111 /¿equiv./l ; insulin 7-7 + 1-1 /tu./ml; glucose 63-3± 1-7 mg/dl; amino nitrogen 3-89 + 0-09 mg/dl. *P < 005; **P < 0005; ***P < 0001. Free
(B)
fatty acid
Glucose r=0-94
F=0-02
-20
25
50
0
1
10
25
50
Ovine placental lactogen (mg) Fig. 2. Relationship between log of the dose'of ovine placental lactogen and the maximum response, expressed as percent of change from baseline concentration. (A) Free fatty acids and insulin; (B) glucose and amino nitrogen. Slopes were calculated by linear regression analysis.
DISCUSSION
Although placental lactogens have been identified in man (Josimovich & McLaren, 1962), monkeys (Shome & Friesen, 1971), and two other subprimate groups, the rodents (Ray, Averill, Lyons & Johnson, 1955; Kelly, Shiu, Robertson & Friesen, 1975) and domestic ruminants (Buttle, Forsyth & Knaggs, 1972; Kelly, Robertson & Friesen, 1974), only the metabolic effects of human placental lactogen have been studied previously. The administra¬ tion of human placental lactogen to human subjects has been demonstrated to cause deterioration in glucose tolerance and an increase in the insulin response to glucose (Beck & Daughaday, 1967; Grumbach et al. 1968). In experiments in vitro, human placental lactogen has been shown to enhance insulin secretion from isolated pancreatic slices (Martin & Friesen, 1969). The effects of human placental lactogen on lipid metabolism are less clear. Prolonged intramuscular administration causes an increase in free fatty acid concentrations (Grumbach, Kaplan, Abrams, Beel & Conte, 1966) but intravenous administration of 4 mg (Berle, Finsterwalder & Apostolakis, 1974) has no effect on free fatty acid concentrations. In vitro, human placental lactogen stimulates both lipolysis (Turtle & Kipnis, 1967; Genazzani, Benuzzi-Badoni & Felber, 1969) and lipogenesis (Felber, Zaragoza, Benuzzi-Badoni & Genazzani, 1972). Our results indicate that ovine, like human placental lactogen, has significant effects on intermediary metabolism although the mechanisms by which these effects are exerted are obscure. After administration of ovine placental lactogen there were acute decreases in both free fatty acid and insulin concentrations. Thereafter, free fatty acid concentrations returned to baseline even though there was a progressive increase in insulin secretion. Plasma glucose and amino nitrogen concentrations began to decline about 1 h after the rise in insulin secretion. Although insulin administration has been demonstrated to cause a decrease in amino nitrogen (Luck, Morrison & Wilbur, 1928), the decrease in amino nitrogen following administration of ovine placental lactogen cannot be attributed entirely to insulin since 5 mg ovine placental lactogen caused a decrease in amino nitrogen concentrations without a change in insulin concentrations. The prolonged effects of ovine placental lactogen administration on insulin, glucose, and amino nitrogen metabolism are striking. Although we have no explanation as yet for these prolonged effects, it is of interest that acute administration of ovine growth hormone and prolactin has also been reported to produce prolonged metabolic effects lasting as long as 24 h (Manns & Boda, 1965; Winkler, Rothgeb, Steele & Altszuler, 1971). It is also of interest that acute administration of ovine placental lactogen caused nearly identical effects in the pregnant and non-pregnant sheep. Studies on chronic administration of ovine placental lactogen in pregnant and non-pregnant sheep are in progress. Grumbach et al. (1968) have postulated that human placental lactogen, by its anabolic and diabetogenic effects, acts metabolically as the growth hormone of pregnancy and permits the foetus to receive an adequate supply of glucose and amino acids. The striking effects of a preparation of ovine placental lactogen free of growth hormone, prolactin, and insulin on carbohydrate, lipid and amino acid metabolism in the pregnant sheep also provide strong support for the thesis that ovine placental lactogen is important in the regulation of intermediary metabolism during pregnancy. The sheep, therefore, provides an excellent animal model for investigation of the physiological role of placental lactogen in maternal and foetal metabolism and the mechanisms by which placental lactogen exerts its metabolic effects.
We thank James B. Sidbury, Harold Lebovitz and George Brumley for helpful discussions, and Elaine Haynes, Rebecca Bowen and Larry Kodack for technical assistance. This work was supported by grants from the National Institutes of Health (HD 07447 and AM 12861) and the National Foundation-March of Dimes (CRBS-297). S.H. is the recipient of a Career Development Award from the U.S. Public Health Service (HD 00065). REFERENCES
Daughaday, W. H. (1967). Human placental lactogen: studies of its acute metabolic effects and Beck, disposition in normal man. Endocrinology 46,103-109. Berle, P., Finsterwalder, E. & Apostolakis, M. (1974). Comparative studies on the effect of human growth hormone, human prolactin and human placental lactogen on lipid metabolism. Hormone and Metabolic Research 6, 347-350. Buttle, H. L., Forsyth, I. A. & Knaggs, G. S. (1972). Plasma prolactin measured by radioimmunoassay and bioassay in pregnant and lactating goats and the occurrence of a placental lactogen. Journal of Endocrinology 53, 483-491. Dole, V. P. (1956). A relation between non-esterified fatty acids in plasma and the metabolism of glucose. Journal of Clinical Investigation 35, 150-154. Felber, J. P., Zaragoza, ., Benuzzi-Badoni, M. & Genazzani, A. R. (1972). The double effect of human chorionic somatomammotropin (HCS) and pregnancy on lipogenesis and lipolysis in the isolated rat epididymal fat pad and fat pad cells. Hormone and Metabolic Research 4, 293-296. Genazzani, A. R., Benuzzi-Badoni, M. & Felber, J. P. (1969). Human chorionic somato-mammotropin (HCSM): lipolytic action of a pure preparation on isolated fat cells. Metabolism 18, 593-598. Goodwin, J. F. (1968). The colorimetrie estimation of plasma amino nitrogen with DNFB. Clinical Chemistry 14, 1080-1090. Grumbach, M. M., Kaplan, S. L., Abrams, C. L., Beel, J. J. & Conte, F. . (1966). Plasma free fatty acid response to the administration of chorionic growth hormone-prolactin. Journal of Clinical Endocrinology and Metabolism 26, 478^182. Grumbach, M. M., Kaplan, S. L., Sciarra, J. J. & Burr, I. M. (1968). Chorionic growth hormone prolactin (CGP): secretion, disposition, biologic activity in man, and postulated function as the 'growth hormone' of the second half of pregnancy. Annals of New York Academy of Sciences 148, 501-531. Handwerger, S., Maurer, W., Barrett, J., Hurley, T. & Fellows, R. E. (1974). Evidence for homology between ovine and human placental lactogens. Endocrine Research Communications 1, 401-413. Hurley, T., Fellows, R. E., Maurer, W. F. & Handwerger, S. (1975). Ovine placental lactogen: structural and functional relationship with primate placental lactogen, growth hormone and prolactin. In Fourth American Peptide Symposium, ed. R. Walter. Ann Arbor: Ann Arbor Science Publishers (in Press). Josimovich, J. B. & McLaren, J. A. (1962). Presence in the human placenta and term serum of a highly lactogenic substance immunologically related to pituitary growth hormone. Endocrinology 71, 209-220. Kelly, P. ., Robertson, H. A. & Friesen, H. G. (1974). Temporal pattern of placental lactogen and pro¬ gesterone secretion in sheep. Nature 248, 435-437. Kelly, P. ., Shiu, R. P. C, Robertson, H. A. & Friesen, H. G. (1975). Characterization of rat chorionic mammotropin. Endocrinology 96, 1187-1195. Luck, J. M., Morrison, G. & Wilbur, L. F. (1928). Effect of insulin on the amino acid content of blood. Journal of Biological Chemistry 77,151-156. Manns, J. G. & Boda, J. M. (1965). Effects of ovine growth hormone and prolactin on blood glucose, serum insulin, plasma nonesterified fatty acids and amino nitrogen in sheep. Endocrinology 76, 1109-1114. Martin, J. M. & Friesen, H. G. (1969). Effect of human placental lactogen on the isolated islets of Langerhans in vitro. Endocrinology 84, 619-621. Morgan, C. R. & Lazarow, A. (1963). Immunoassay of insulin: two antibody system. Plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 12, 115-126. Ray, E. W., Averill, S. C, Lyons, W. R. & Johnson, R. E. (1955). Rat placental hormonal activities corre¬ sponding to those of pituitary mammotropin. Endocrinology 56, 359-373. Shome, . & Friesen, H. G. (1971). Purification and characterization of monkey placental lactogen. Endocrinology 89, 631-641. Turtle, J. R. & Kipnis, D. M. (1967). The lipolytic action of human placental lactogen on isolated fat cells. Biochimica et Biophysica Acta 144, 583-594. Winkler, B., Rothgeb, L, Steele, R. & Altszuler, N. (1971). Effect of ovine prolactin administration on free fatty acid metabolism in the normal dog. Endocrinology 88, 1349-1352. P. &
