The Effects of Insulin and Growth Hormone on the Release of Somatomedin by the Isolated Rat Liver WILLIAM H. DAUGHADAY, LAWRENCE S. PHILLIPS,* AND MARY C. MUELLER Department of Medicine, Metabolism Division, and the Diabetes Center, Washington University School of Medicine, St. Louis, Missouri 63110 insulin was without significant effect. The possible inter-relationship between insulin and growth hormone in the regulation of somatomedin release was studied with a dose of bGH of 250 ng/ml which had previously been shown to be insufficient by itself to stimulate somatomedin release. When added to a medium containing 1000 fx\J/m\ of insulin, this dose of bGH did not significantly stimulate somatomedin release beyond that obtained with insulin alone. However, when 250 ng/ml was added to a medium containing 100 /xU/ml
ABSTRACT. Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 /xU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 /xU/ml of
T
HE ABILITY of bovine growth hormone, 10 /Ltg/ml, added to perfusates of isolated normal rat livers to stimulate the release of somatomedin-like activity was reported by McConaghey and Sledge (1) and by McConaghey (2). In these experiments, the somatomedin-like activity was assayed on hypophysectomized rat cartilage. Francis and Hill (3) have recently demonstrated that ovine prolactin in concentrations as low as 50 ng/ml was capable of stimulating the re-
Received September 15, 1975. Presented in part at the 3rd International Symposium on Growth Hormone and Related Peptides, Milan, Italy, September, 1975. Supported in part by Research Grants no. AM01526 and AM17904 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland. * Trainee in Metabolism, supported by Training Grant no. AM05027 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland. Present address: Division of Endocrinology, Northwestern University Medical School, Chicago, Illinois 60611.
insulin, a significant stimulation of somatomedin
release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels. (Endocrinology 98: 1214, 1976)
lease of somatomedin-like activity from perfused normal rat liver. We have investigated the release of somatomedin in a somewhat different liver perfusion system employing a higher flow rate, which maintained normal oxygenation of the liver as demonstrated by lactate: pyruvate ratios within the normal range for rat plasma (4). The experimental design included recirculation of perfusion medium for 30 minute periods for 3 hours. With this system we demonstrated that the basal release of somatomedin was greater in normal than in hypophysectomized male rats. In vivo treatment of the hypophysectomized rat liver donors resulted in an increase in the somatomedin activity of the perfusates. We observed at best a marginal stimulation of the release of somatomedin by livers from normal rats when 25 fig of bGH was added to our perfusion medium. The addition of this concentration of bGH to perfusates of livers from hypox livers signifi-
1214
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INSULIN AND HEPATIC SOMATOMEDIN RELEASE cantly stimulated the release of somatomedin (Sm) compared with that of control livers not perfused with bGH at the same time period. Two hundred and fifty ng of bGH/ml of perfusate appeared to be without effect. In the present investigation we have examined the possible stimulation of Sm release from livers when insulin was added to the perfusion medium and the possible interaction between insulin and GH in regulating the release of Sm from the isolated rat livers. Materials and Methods The livers from 85 to 110 g hypophysectomized male Sprague-Dawley rats were removed rapidly from the body and perfused by way of the portal vein in an oxygenated recirculating system. The details of our apparatus and methods have been described (4). The perfusion medium was a single lot of Waymouth's MB 752/1 (Grand Island Biological Company) to which was added 1% bovine serum albumin (Fraction V, Sigma Chemical Company). Perfusions were carried out for 3 hours with a change of medium (40 ml) every 30 minutes. Hormone additions were always made after the first of six 30 minute perfusion periods. Bovine growth hormone (NIH-GH-B17) was added to the perfusion medium in concentrations of 25 /ug/ml (high bGH) and 0.25 /xg/ml (low bGH). Insulin (Iletin, Eli Lilly) was added in concentrations of 1000 juU/ml (high insulin) and 100 /u-U/ml (low insulin). In certain experiments in which insulin was added to the perfusion medium the concentration of insulin in the medium was measured before and after perfusion of the liver by radioimmunoassay (5). Somatomedin activity was detected by measurement of the stimulation of sulfate uptake by isolated segments of rat costal cartilage (6). Sixty small segments of costal cartilage from a single hypophysectomized rat were distributed among 12 incubation tubes containing 0.4 ml of the Waymouth's perfusion medium or 0.4 ml of the perfusates of livers at the various time periods. 1 All specimens from the same experi1
Studies of serial dilutions of perfusates of normal rat liver indicated that the response of sulfate uptake
1215
ment were incubated with five costal cartilage segments from the same rat. The unperfused medium was incubated with 10 cartilage segments to improve replicability. A supplement of 50 /A containing 50 units of penicillin, 200 fxg of streptomycin, 5 /ug of amphotericin, and 10 /jig of polymyxin was added to each tube. After 20 hours of preincubation, 50 /xl containing approximately 1 fid [35S]sulfate was added to each tube, and the incubation was continued for an additional 5 hours. The cartilage segments were then washed, dried, and weighed. They were dissolved in KOH prior to scintillation counting. The results are expressed as the ratio of cpm of the perfused buffer divided by the unperfused buffer. In perfusions with added hormones, the buffer control also contained these hormones. While this calculation minimizes rat to rat and day to day variance the results are not directly related to Sm concentration but will be referred to as Sm activity or per cent of stimulation of sulfate uptake. The results have been analyzed for corresponding periods by Student's t test (two-tailed) and by a three-way analysis of variance. The calculation of sulfate uptake presumes that no change in the sulfate pool of the medium occurred during incubation. This has been checked by measuring the inorganic sulfate (7) in the perfusion medium before and after liver perfusion in each experimental condition during the 6 changes of medium. The rise of medium sulfate concentration during the perfusion of hypox livers without added hormone was negligible. When insulin, 1000 fx\J/m\, was added, the rise was 13%, and when GH, 25 /ag/ml, was added this amounted to 9%. No correction was introduced in the calculations for these relatively slight changes in medium sulfate pool. Results
The addition of insulin and bGH to the unperfused medium in the concentrations was roughly proportional to the logarithm of the concentration of medium in the assay system. The stimulation of sulfate observed when perfusates of hypox rat liver were added to our test hypophysectomized rat cartilage was about half of that which resulted when perfusates of normal rat liver were added. Thus, we were operating in a favorable portion of the doseresponse curve.
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Endo • 1976 Vol 98 • No 5
DAUGHADAY, PHILLIPS AND MUELLER
1216
TABLE 1. Effect of hormone addition to unperfused buffer on uptake of sulfate by hypox rat cartilage
Addition
N
fig SO4/IOO mg
Buffer plus hormone fig SCyiOO mg
bGH .25 jug/ml bGH 25 /xg/ml Insulin 100 /xU/ml Insulin 1,000 /xU/ml Insulin 100 /xU/ml + bGH .25 /xg/ml Insulin 1,000 /xU/ml + bGH .25 /xg/ml
11 10 10 10 10 10
1.80 1.50 1.33 1.86 1.47 1.49
1.88 1.64 1.44 1.82 1.37 1.47
Buffer
Mean difference ± SE .09 dt .14 dt .11 dt - . 0 4 dt .10 dt - . 0 2 dt
.06 .09 .09 .19 .08 .11
Per cent stimulation 5.3 dt 4.2 9.7 d: 13 7.8 dt 12 - 2 . 2 dt 8.6 - 5 . 7 dt 5.4 7.2 db 8.9
The mean uptakes of sulfate in unperfused buffer and unperfused buffer plus the indicated hormone are tabulated. The mean difference of uptake ± SE between the two uptakes in each incubation is provided. The per cent stimulation is mean of the calculated value for each incubation ± SE.
used in these experiments had insignificant stimulatory effects on sulfate uptake (Table 1).
The results of measurement of Sm activity in perfusates of hypox liver are all presented in Table 2. When no hormone
additions were made to the perfusion medium there was a progressive decline in apparent Sm activity during the period of perfusion (Group A). bGH in a concentration of 25 /xg/ml significantly increased sulfate uptake during periods 3 to 6 when
TABLE 2. Per cent stimulation of sulfate uptake compared with unperfused medium (Mean ± SE)
Control period 1
2
3
4
5
6
A 0
72 ± 10
62 ± 10
45 ± 9
33 ± 8
25 ± 11
20 ± 10
B
78 ± 13
116 ±27
82 ± 13*
114 ± 431
88 ± 21t
77 ± 27*
64 ± 11
62 ± 8
58 ± 7
32 ± 9
27 ± 9
24 ± 11
78 ± 14
101 ± 12f
98 ± 15*
89 ± 15t
88 ± 12f
77 ± 12t
72 ± 13
51 ±18
54 ± 12
55 ±10
51 ± 13
41 ± 10
78 ± 11
92 ± 16*
92 ± 17*
71 ± 13*
65 ± 14*
61 ± 16*
85 ± 12
82 ±12
75 ± 12*
74 ± 10f
71 ± 14*
52 ± 13
Additions to medium
C D E F
n = 16*| bGH25fig/m\\ n=5 bGH 0.25 fig/mlt n = 10 Insulin 1,000 /xU/ml n = 10 Insulin 100 tiU/ml n=10 Insulin 1,000 /xU/ml + bGH 0.25
Experimental periods
/ttg/ml
n = 10 G Insulin 100 /xU/ml + bGH 0.25 /xg/ml n = 10
Analysis of variance Treatment effect difference from group A (periods 2 to 6) B C D E F G
P < 0.001 NS P < 0.001 (also P < 0.001 from E) NS P < 0.015 (also P < 0.05 from C but NS from D) P < 0.05 (also P < 0.05 from C but NS from E)
Time effect
Interactions between treatment and time effects
P < 0.001 P < 0.001 NS P < 0.01 P < 0.001 P
ABSTRACT. Livers from hypophysectomized (hypox) rats were perfused with oxygenated Waymouth's medium in a system which permitted continuous recirculation for separate 30 minute periods after which fresh medium was supplied. In most experiments 6 changes of medium were carried out over a 3 hour period. The somatomedin activity of each perfusate was determined by measuring its ability to stimulate sulfate uptake in hypox rat cartilage in vitro. For comparison between experiments the results are expressed as the per cent stimulation of sulfate uptake by the perfusate compared with the unperfused buffer. Without hormonal additions there was a progressive fall in the release of somatomedin activity during the 6 periods of study. When compared with the results without hormone, the addition of 1000 /xU/ml of insulin per ml of medium during the 2nd to 6th period led to a significant increase in perfusate somatomedin activity at all periods. The addition of 100 /xU/ml of
T
HE ABILITY of bovine growth hormone, 10 /Ltg/ml, added to perfusates of isolated normal rat livers to stimulate the release of somatomedin-like activity was reported by McConaghey and Sledge (1) and by McConaghey (2). In these experiments, the somatomedin-like activity was assayed on hypophysectomized rat cartilage. Francis and Hill (3) have recently demonstrated that ovine prolactin in concentrations as low as 50 ng/ml was capable of stimulating the re-
Received September 15, 1975. Presented in part at the 3rd International Symposium on Growth Hormone and Related Peptides, Milan, Italy, September, 1975. Supported in part by Research Grants no. AM01526 and AM17904 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland. * Trainee in Metabolism, supported by Training Grant no. AM05027 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, Bethesda, Maryland. Present address: Division of Endocrinology, Northwestern University Medical School, Chicago, Illinois 60611.
insulin, a significant stimulation of somatomedin
release was observed while the addition of each hormone separately was without significant effect. These results support the hypothesis that insulin shares with GH the regulation of somatomedin release by the liver. Differences in insulin concentration may explain some clinical situations in which somatomedin concentrations cannot be correlated with GH levels. (Endocrinology 98: 1214, 1976)
lease of somatomedin-like activity from perfused normal rat liver. We have investigated the release of somatomedin in a somewhat different liver perfusion system employing a higher flow rate, which maintained normal oxygenation of the liver as demonstrated by lactate: pyruvate ratios within the normal range for rat plasma (4). The experimental design included recirculation of perfusion medium for 30 minute periods for 3 hours. With this system we demonstrated that the basal release of somatomedin was greater in normal than in hypophysectomized male rats. In vivo treatment of the hypophysectomized rat liver donors resulted in an increase in the somatomedin activity of the perfusates. We observed at best a marginal stimulation of the release of somatomedin by livers from normal rats when 25 fig of bGH was added to our perfusion medium. The addition of this concentration of bGH to perfusates of livers from hypox livers signifi-
1214
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INSULIN AND HEPATIC SOMATOMEDIN RELEASE cantly stimulated the release of somatomedin (Sm) compared with that of control livers not perfused with bGH at the same time period. Two hundred and fifty ng of bGH/ml of perfusate appeared to be without effect. In the present investigation we have examined the possible stimulation of Sm release from livers when insulin was added to the perfusion medium and the possible interaction between insulin and GH in regulating the release of Sm from the isolated rat livers. Materials and Methods The livers from 85 to 110 g hypophysectomized male Sprague-Dawley rats were removed rapidly from the body and perfused by way of the portal vein in an oxygenated recirculating system. The details of our apparatus and methods have been described (4). The perfusion medium was a single lot of Waymouth's MB 752/1 (Grand Island Biological Company) to which was added 1% bovine serum albumin (Fraction V, Sigma Chemical Company). Perfusions were carried out for 3 hours with a change of medium (40 ml) every 30 minutes. Hormone additions were always made after the first of six 30 minute perfusion periods. Bovine growth hormone (NIH-GH-B17) was added to the perfusion medium in concentrations of 25 /ug/ml (high bGH) and 0.25 /xg/ml (low bGH). Insulin (Iletin, Eli Lilly) was added in concentrations of 1000 juU/ml (high insulin) and 100 /u-U/ml (low insulin). In certain experiments in which insulin was added to the perfusion medium the concentration of insulin in the medium was measured before and after perfusion of the liver by radioimmunoassay (5). Somatomedin activity was detected by measurement of the stimulation of sulfate uptake by isolated segments of rat costal cartilage (6). Sixty small segments of costal cartilage from a single hypophysectomized rat were distributed among 12 incubation tubes containing 0.4 ml of the Waymouth's perfusion medium or 0.4 ml of the perfusates of livers at the various time periods. 1 All specimens from the same experi1
Studies of serial dilutions of perfusates of normal rat liver indicated that the response of sulfate uptake
1215
ment were incubated with five costal cartilage segments from the same rat. The unperfused medium was incubated with 10 cartilage segments to improve replicability. A supplement of 50 /A containing 50 units of penicillin, 200 fxg of streptomycin, 5 /ug of amphotericin, and 10 /jig of polymyxin was added to each tube. After 20 hours of preincubation, 50 /xl containing approximately 1 fid [35S]sulfate was added to each tube, and the incubation was continued for an additional 5 hours. The cartilage segments were then washed, dried, and weighed. They were dissolved in KOH prior to scintillation counting. The results are expressed as the ratio of cpm of the perfused buffer divided by the unperfused buffer. In perfusions with added hormones, the buffer control also contained these hormones. While this calculation minimizes rat to rat and day to day variance the results are not directly related to Sm concentration but will be referred to as Sm activity or per cent of stimulation of sulfate uptake. The results have been analyzed for corresponding periods by Student's t test (two-tailed) and by a three-way analysis of variance. The calculation of sulfate uptake presumes that no change in the sulfate pool of the medium occurred during incubation. This has been checked by measuring the inorganic sulfate (7) in the perfusion medium before and after liver perfusion in each experimental condition during the 6 changes of medium. The rise of medium sulfate concentration during the perfusion of hypox livers without added hormone was negligible. When insulin, 1000 fx\J/m\, was added, the rise was 13%, and when GH, 25 /ag/ml, was added this amounted to 9%. No correction was introduced in the calculations for these relatively slight changes in medium sulfate pool. Results
The addition of insulin and bGH to the unperfused medium in the concentrations was roughly proportional to the logarithm of the concentration of medium in the assay system. The stimulation of sulfate observed when perfusates of hypox rat liver were added to our test hypophysectomized rat cartilage was about half of that which resulted when perfusates of normal rat liver were added. Thus, we were operating in a favorable portion of the doseresponse curve.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 14 December 2014. at 16:52 For personal use only. No other uses without permission. . All rights reserved.
Endo • 1976 Vol 98 • No 5
DAUGHADAY, PHILLIPS AND MUELLER
1216
TABLE 1. Effect of hormone addition to unperfused buffer on uptake of sulfate by hypox rat cartilage
Addition
N
fig SO4/IOO mg
Buffer plus hormone fig SCyiOO mg
bGH .25 jug/ml bGH 25 /xg/ml Insulin 100 /xU/ml Insulin 1,000 /xU/ml Insulin 100 /xU/ml + bGH .25 /xg/ml Insulin 1,000 /xU/ml + bGH .25 /xg/ml
11 10 10 10 10 10
1.80 1.50 1.33 1.86 1.47 1.49
1.88 1.64 1.44 1.82 1.37 1.47
Buffer
Mean difference ± SE .09 dt .14 dt .11 dt - . 0 4 dt .10 dt - . 0 2 dt
.06 .09 .09 .19 .08 .11
Per cent stimulation 5.3 dt 4.2 9.7 d: 13 7.8 dt 12 - 2 . 2 dt 8.6 - 5 . 7 dt 5.4 7.2 db 8.9
The mean uptakes of sulfate in unperfused buffer and unperfused buffer plus the indicated hormone are tabulated. The mean difference of uptake ± SE between the two uptakes in each incubation is provided. The per cent stimulation is mean of the calculated value for each incubation ± SE.
used in these experiments had insignificant stimulatory effects on sulfate uptake (Table 1).
The results of measurement of Sm activity in perfusates of hypox liver are all presented in Table 2. When no hormone
additions were made to the perfusion medium there was a progressive decline in apparent Sm activity during the period of perfusion (Group A). bGH in a concentration of 25 /xg/ml significantly increased sulfate uptake during periods 3 to 6 when
TABLE 2. Per cent stimulation of sulfate uptake compared with unperfused medium (Mean ± SE)
Control period 1
2
3
4
5
6
A 0
72 ± 10
62 ± 10
45 ± 9
33 ± 8
25 ± 11
20 ± 10
B
78 ± 13
116 ±27
82 ± 13*
114 ± 431
88 ± 21t
77 ± 27*
64 ± 11
62 ± 8
58 ± 7
32 ± 9
27 ± 9
24 ± 11
78 ± 14
101 ± 12f
98 ± 15*
89 ± 15t
88 ± 12f
77 ± 12t
72 ± 13
51 ±18
54 ± 12
55 ±10
51 ± 13
41 ± 10
78 ± 11
92 ± 16*
92 ± 17*
71 ± 13*
65 ± 14*
61 ± 16*
85 ± 12
82 ±12
75 ± 12*
74 ± 10f
71 ± 14*
52 ± 13
Additions to medium
C D E F
n = 16*| bGH25fig/m\\ n=5 bGH 0.25 fig/mlt n = 10 Insulin 1,000 /xU/ml n = 10 Insulin 100 tiU/ml n=10 Insulin 1,000 /xU/ml + bGH 0.25
Experimental periods
/ttg/ml
n = 10 G Insulin 100 /xU/ml + bGH 0.25 /xg/ml n = 10
Analysis of variance Treatment effect difference from group A (periods 2 to 6) B C D E F G
P < 0.001 NS P < 0.001 (also P < 0.001 from E) NS P < 0.015 (also P < 0.05 from C but NS from D) P < 0.05 (also P < 0.05 from C but NS from E)
Time effect
Interactions between treatment and time effects
P < 0.001 P < 0.001 NS P < 0.01 P < 0.001 P
