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Human somatomedin A and longitudinal bone growth in the hypophysectomized rat
Molecular and Cellular Endocrinology, 6 (1977) 217-221 0 Elsevier/North-Holland Scientific Publishers Ltd.
HUMAN SOMATOMEDIN A AND LONGITUDINAL HYPOPHYSECTOMIZED
K.G. THORNGREN, Department Polypeptide Received
BONE GROWTH IN THE
RAT
L.I. HANSSON, L. FRYKLUND
* and H. SIEVERTSSON
*
of Orthopaedic Surgery, University of Lund, Lund, and * AB KABI, The Recip Laboratory, Stockholm, Sweden
5 April 1976; accepted
25 June 1976
A somatomedin A preparation, when given at total doses of 14 and 70 U did not increase the longitudinal bone growth in hypophysectomized rats. Growth hormone (WHO) significantly increased the longitudinal bone growth. Keywords:
somatomedin;
growth
hormone;
bone; growth;
rat.
It is well known that growth hormone stimulates longitudinal bone growth in the hypophysectomized rat (for review, see Asling and Evans, 1956; Urist, 1972; Thorngren and Hansson, 1974a). In recent years somatomedin has been proposed to be mediator for the growth effect of growth hormone, and different types of somatomedins have been discovered (for review, see Luft and Hall, 1975). Up to the present, the supply of pure somatomedins has been insufficient for in vivo investigations. The majority of the reports on the physiological significance of somatomedins have been in vitro studies utilizing various animal tissues. Recently partially purified somatomedin preparations have been given in vivo to hypophysectomized rats (Uthne, 1975; Fryklund et al., 1975) where certain in vivo effects can be observed. For the further clarification of the physiological role of the somatomedins, the demonstration of their eventual in vivo effects are of great importance. In order to investigate the properties of new growth-promoting substances it is necessary to determine the growth stimulation by bioassay. Thorngren and Hansson (1974a,b,c) have developed a very sensitive and accurate bioassay for measuring longitudinal bone growth in hypophysectomized rats using tetracycline as an intravital marker. In the present investigation somatomedin A was given at two dose levels and the longitudinal bone growth and the body-weight were determined.
MATERIAL
AND METHODS
A standardized bioassay procedure was used (Thorngren and Hansson, 1974a,b,c). Hypophysectomized female rats were given one daily S.C. hormone 217
218
K.G. Thorngren
et al.
injection in association with L-thyroxine for 5 days followed by a withdrawal period of 10 days, as described in detail elsewhere (Thorngren and Hansson, 1974~). The longitudinal bone growth was determined microscopically using tetracycline as intravital marker (Thorngren and Hansson, 1974a,b,c). A partially purified human somatomedin A preparation isolated according to Fryklund et al. (1974) was used. This contained about 200 U/mg protein, as determined by the chickembryo bioassay (Hall, 1970). Also, the radioreceptor assay on human placental membranes (Jail et al., 1974; Takano et al., 1975) showed similar values when this preparation was tested at five dose levels against a somatomedin A standard containing by definition 200 U/mg (h = 0.04, fiducial limits = 95%). Growth hormone (1st International Standard for GH) was obtained from the National Institute for Biological Standards and Control, London, U.K. By definition this preparation contains 1 IU growth hormone per mg.
RESULTS See table 1. Animals given growth hormone showed a significant increase in longitudinal bone growth and body-weight during the administration period. Then the body weight decreased somewhat during the withdrawal period. At the tested doselevels no significant difference in bone growth was found between the controls and animals given somatomedin A. Animals given somatomedin A showed a minor but statistically insignificant weight increase at the end of the withdrawal period. The
OTC u t
600
CONTROLS THYR
’ SOMATOMEDIN THYR
2.8 u
14u
A
WHO- GH
20/&I
8Opg
+BUFFER Fig. 1. Effect of somatomedin A and growth hormone nal bone growth of proximal tibia in hypophysectomized
(daily rats.
doses) on accumulated
longitudi-
daily dose
111 f 9
4
P < 0.001. P > 0.05.
13
71 * 22
4
94*
392 * 19
7
5
123 + I 288 * 21
Uncorrected
Act. growth
9 6
* By i-test versus control, ** By i-test versus control,
Thyroxine (20 pg/kg) WHO-GH (20 fig) + Thyroxine WHO-GH (80 /.& + Thyroxine Somatomedin buffer (0.5 ml) +Thyroxine Somatomedin (2.8 U) + Thyroxine Somatomedin (14 U) + Thyroxine
(no.)
Animals
172 f 2 165 f 6
175 + 2 176 2 8
159 + 5 157 f 6
157 f 4 162 + 3
422 + 17 * 112+
116+99**
132*10**
21
173 +_4
171 I3
change days
3*1**
3*1**
-2 * 4
12+2*
15 + 1*
4+1
15-20
Weight
15 days
(g)
postop.
Body weight
17822
Cartilage width (pm)
165 i- 3
(pm)
140 * 10 314 + 15 *
Corrected
in length
change
179 + 3
177 + 3
‘63+9
5+3**
7+3**
4?5
days
2 **
-4+
184+
3
-2i: 3 -4+-l**
182 f 2 187 + 3
20-30
Weight
20 days postop.
..~.
2
184~
1
185 f 5
167 + 4
1805
._~ 179 f 3 183 + 3
postop.
30 days
-09
somatomedin A and growth hormone (GH; 1st International Standard for Growth Hormone from WHO). Female rats hypophysectoage 60 days. Administration of the hormones in association with L-thyroxine on days 15-19 postoperatively. Accumulated growth in proximal tibia 15-30 days postoperatively. Width of growth plate of proximal tibia 30 days postoperatively. Bodyweight at various postintervals. Values are given as mean 2 S.E.M.
_____-.-..
Hormone
Table 1 Effect of mized at length of operative
2 P
B 3 2 ;
220
K. C. Tlzorngren et al.
cartilage width had decreased to non-stimulated values because of the withdrawal period used, indicating that no further longitudinal bone growth could bc expected by the given administration. All the eventua~y produced cartilage cells have then been transformed into bone by the e~dochondr~ growth process.
DISCUSSION Recent studies have revealed different growth ho~one-dependent polypeptide hormones in the control of cell growth (for review, see Luft and Hall, 1975). Four such factors have been purified from serum: somatomedins A, B and C and NSILA-S. Of these somatomedin A, C and NSILAS have many target tissues in common (Hall et al.,, 1975). Somatomedin A has already shown biological action when tested in in vitro systems. Thus, it stimulates the sulfate and thymidine uptake in cartilage of rat and chicken (Hall, 1972; Hall et al., 1975). It also exerts insulin-like activity on rat adipose tissue, stimulates the incorporation of amino acids into muscle protein, and glucose uptake into muscle. Somatomedin A also binds to liver membrane and human placenta (Hall et al., 1975). Very few studies concerning the effect of somatomedins in vivo have appeared (Uthne, 1975). Limited availability of the purified hormone has been a great restriction. Somatomedin is extracted from human plasma and purified. This procedure is tedious and demands large initial quantities (Sievertsson et al., 1975). Thus, for the present study a limited amount of somatomedin A was obtained, restricting the number of dose levels and animals in the bioassay. In an earlier study, somatomedin A when given to hypophysectomi~ed rats significantly increased tibial cartilage width by 30% whereas no signi~cant effect on total body-weight was registered in the same animals (Uthne, 1975). The material used in that study, however, had been taken from a gel-filtration on Sephadex G-50 after the acidethanol extraction. According to Uthne (1975), the possibility could not therefore be excluded that the stimulatory effect found on the tibia1 cartilage width might be attributed to a combined effect of several factors, some of them not necessarily carrying somatomedin A activity. The somatomedin A dose then was 15 U/rat/day given over 5 days, which amounts to a total dose of 75 U. tn the present study, the material had been further fractionated by electrophoresis at pH 7.5 and 5.0 and is therefore not comparable. In the present investigation no stimulation by somatomedin A on the longitudinal bone growth was found for total doses of 14 and 70 U. This might be due to the dose levels being too low, or to some as yet. unknown factor, such as rapid inactivation or excretion in vivo or species specificity. Also, contaminating peptides in the preparation may have had a negative influence on the effect of the purified somatomedin A. Recent in viva studies with somatomedin A given to hypophysectomized rats have shown a significa.nt stimulatory influence on the sulfate incorporation into
Sohatomedin
and bone gpowth
221
skin (Fryklund et al., 1976) and costal cartilage (unpublished). NO definite effect, however, was found on the longitudinal bone growth in the present study, which might indicate a previously unr~co~n~ed specificity for the somatomedin A effect in different tissues. The present results contribute some preliminary findings to the scarce information on eventual in vivo effects of the somatomedins. Further investigations with higher dose levels and more purified preparations would be of great interest.
ACKNOWLEDGEMENTS The investigation was supported by the Faculty of Medicine, University of Lund and was carried out within a research organization sponsored by the Swedish Medical Research Council (Project 17 X-203 2).
REFERENCES Asling, C.W. and Evans, H.M. (1956) In: The Biochemistry and Physiology of Bone, Ed.: G.H. Bourne, (Academic Press, New York) pp. 671-703. Fryklund, L., Uthne, K. and Sievertsson, H. (1974) Biochem. Biophys. Res. ~ommun. 61, 957-962.
Fryklund, L., Skottner, A. and Sievertsson, H. (1976) In: Proc. 3rd Int. Congr. Growth Hormone (Excerpta Medica, Amsterdam) in press. HaU, K. (1970) Acta Endocrinol. (Kbh.) 63, 338-350. Ha&K. (1972) Acta Endocrinol. (Kbh.) suppl. 163. Hall, K., Takano. K., Fryktund, L. (1974) J. Clin. Endocrinol. 39,973-976. Hall, K., Takano., K., Fryklund, L. and Sievertsson, H. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. HaII (Academic Press, New York) pp. 19-46. Luft, R. and Hall, K. (1975) (Eds.) Somatomedins and Some Other Growth Factors. Adv. Metab. Dis., Vol. 8 (Academic Press, New York). Siovertsson, H., Ftyklund, L. Uthne, K., Hall, K. and Westermark, B. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. Hall (Academic Press, New York) pp. 47-60. Takano, K., HalI, K., Fryklund, L., Holmgren, A., Sievertsson, H. and Uthne, K, (1975) Acta Endocrinol. (Kbh.) 80, 1. Thorngren, K.-G. and Hansson, L.I. (1974a) Acta Endocrinol. (Kbh.) 75,653-668. Thorngren, K.C. and Hansson, L.I. (1974b) Acta Endocrinol. (Kbh.) 75,669-682. Thorngren, K.G. and Hansson. L.I. (1974~) Acta Endocrinol. (Kbh.) 76,35-52. Urist, M.R. (1972) In: The Biochemistry and Physiology of Bone, Vol. II. Ed.: G.H. Bourne (Academic Press, New York) pp. 155-195. Uthne, K. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. Halt. (Academic Press, New York) pp. 11.5-126.
HUMAN SOMATOMEDIN A AND LONGITUDINAL HYPOPHYSECTOMIZED
K.G. THORNGREN, Department Polypeptide Received
BONE GROWTH IN THE
RAT
L.I. HANSSON, L. FRYKLUND
* and H. SIEVERTSSON
*
of Orthopaedic Surgery, University of Lund, Lund, and * AB KABI, The Recip Laboratory, Stockholm, Sweden
5 April 1976; accepted
25 June 1976
A somatomedin A preparation, when given at total doses of 14 and 70 U did not increase the longitudinal bone growth in hypophysectomized rats. Growth hormone (WHO) significantly increased the longitudinal bone growth. Keywords:
somatomedin;
growth
hormone;
bone; growth;
rat.
It is well known that growth hormone stimulates longitudinal bone growth in the hypophysectomized rat (for review, see Asling and Evans, 1956; Urist, 1972; Thorngren and Hansson, 1974a). In recent years somatomedin has been proposed to be mediator for the growth effect of growth hormone, and different types of somatomedins have been discovered (for review, see Luft and Hall, 1975). Up to the present, the supply of pure somatomedins has been insufficient for in vivo investigations. The majority of the reports on the physiological significance of somatomedins have been in vitro studies utilizing various animal tissues. Recently partially purified somatomedin preparations have been given in vivo to hypophysectomized rats (Uthne, 1975; Fryklund et al., 1975) where certain in vivo effects can be observed. For the further clarification of the physiological role of the somatomedins, the demonstration of their eventual in vivo effects are of great importance. In order to investigate the properties of new growth-promoting substances it is necessary to determine the growth stimulation by bioassay. Thorngren and Hansson (1974a,b,c) have developed a very sensitive and accurate bioassay for measuring longitudinal bone growth in hypophysectomized rats using tetracycline as an intravital marker. In the present investigation somatomedin A was given at two dose levels and the longitudinal bone growth and the body-weight were determined.
MATERIAL
AND METHODS
A standardized bioassay procedure was used (Thorngren and Hansson, 1974a,b,c). Hypophysectomized female rats were given one daily S.C. hormone 217
218
K.G. Thorngren
et al.
injection in association with L-thyroxine for 5 days followed by a withdrawal period of 10 days, as described in detail elsewhere (Thorngren and Hansson, 1974~). The longitudinal bone growth was determined microscopically using tetracycline as intravital marker (Thorngren and Hansson, 1974a,b,c). A partially purified human somatomedin A preparation isolated according to Fryklund et al. (1974) was used. This contained about 200 U/mg protein, as determined by the chickembryo bioassay (Hall, 1970). Also, the radioreceptor assay on human placental membranes (Jail et al., 1974; Takano et al., 1975) showed similar values when this preparation was tested at five dose levels against a somatomedin A standard containing by definition 200 U/mg (h = 0.04, fiducial limits = 95%). Growth hormone (1st International Standard for GH) was obtained from the National Institute for Biological Standards and Control, London, U.K. By definition this preparation contains 1 IU growth hormone per mg.
RESULTS See table 1. Animals given growth hormone showed a significant increase in longitudinal bone growth and body-weight during the administration period. Then the body weight decreased somewhat during the withdrawal period. At the tested doselevels no significant difference in bone growth was found between the controls and animals given somatomedin A. Animals given somatomedin A showed a minor but statistically insignificant weight increase at the end of the withdrawal period. The
OTC u t
600
CONTROLS THYR
’ SOMATOMEDIN THYR
2.8 u
14u
A
WHO- GH
20/&I
8Opg
+BUFFER Fig. 1. Effect of somatomedin A and growth hormone nal bone growth of proximal tibia in hypophysectomized
(daily rats.
doses) on accumulated
longitudi-
daily dose
111 f 9
4
P < 0.001. P > 0.05.
13
71 * 22
4
94*
392 * 19
7
5
123 + I 288 * 21
Uncorrected
Act. growth
9 6
* By i-test versus control, ** By i-test versus control,
Thyroxine (20 pg/kg) WHO-GH (20 fig) + Thyroxine WHO-GH (80 /.& + Thyroxine Somatomedin buffer (0.5 ml) +Thyroxine Somatomedin (2.8 U) + Thyroxine Somatomedin (14 U) + Thyroxine
(no.)
Animals
172 f 2 165 f 6
175 + 2 176 2 8
159 + 5 157 f 6
157 f 4 162 + 3
422 + 17 * 112+
116+99**
132*10**
21
173 +_4
171 I3
change days
3*1**
3*1**
-2 * 4
12+2*
15 + 1*
4+1
15-20
Weight
15 days
(g)
postop.
Body weight
17822
Cartilage width (pm)
165 i- 3
(pm)
140 * 10 314 + 15 *
Corrected
in length
change
179 + 3
177 + 3
‘63+9
5+3**
7+3**
4?5
days
2 **
-4+
184+
3
-2i: 3 -4+-l**
182 f 2 187 + 3
20-30
Weight
20 days postop.
..~.
2
184~
1
185 f 5
167 + 4
1805
._~ 179 f 3 183 + 3
postop.
30 days
-09
somatomedin A and growth hormone (GH; 1st International Standard for Growth Hormone from WHO). Female rats hypophysectoage 60 days. Administration of the hormones in association with L-thyroxine on days 15-19 postoperatively. Accumulated growth in proximal tibia 15-30 days postoperatively. Width of growth plate of proximal tibia 30 days postoperatively. Bodyweight at various postintervals. Values are given as mean 2 S.E.M.
_____-.-..
Hormone
Table 1 Effect of mized at length of operative
2 P
B 3 2 ;
220
K. C. Tlzorngren et al.
cartilage width had decreased to non-stimulated values because of the withdrawal period used, indicating that no further longitudinal bone growth could bc expected by the given administration. All the eventua~y produced cartilage cells have then been transformed into bone by the e~dochondr~ growth process.
DISCUSSION Recent studies have revealed different growth ho~one-dependent polypeptide hormones in the control of cell growth (for review, see Luft and Hall, 1975). Four such factors have been purified from serum: somatomedins A, B and C and NSILA-S. Of these somatomedin A, C and NSILAS have many target tissues in common (Hall et al.,, 1975). Somatomedin A has already shown biological action when tested in in vitro systems. Thus, it stimulates the sulfate and thymidine uptake in cartilage of rat and chicken (Hall, 1972; Hall et al., 1975). It also exerts insulin-like activity on rat adipose tissue, stimulates the incorporation of amino acids into muscle protein, and glucose uptake into muscle. Somatomedin A also binds to liver membrane and human placenta (Hall et al., 1975). Very few studies concerning the effect of somatomedins in vivo have appeared (Uthne, 1975). Limited availability of the purified hormone has been a great restriction. Somatomedin is extracted from human plasma and purified. This procedure is tedious and demands large initial quantities (Sievertsson et al., 1975). Thus, for the present study a limited amount of somatomedin A was obtained, restricting the number of dose levels and animals in the bioassay. In an earlier study, somatomedin A when given to hypophysectomi~ed rats significantly increased tibial cartilage width by 30% whereas no signi~cant effect on total body-weight was registered in the same animals (Uthne, 1975). The material used in that study, however, had been taken from a gel-filtration on Sephadex G-50 after the acidethanol extraction. According to Uthne (1975), the possibility could not therefore be excluded that the stimulatory effect found on the tibia1 cartilage width might be attributed to a combined effect of several factors, some of them not necessarily carrying somatomedin A activity. The somatomedin A dose then was 15 U/rat/day given over 5 days, which amounts to a total dose of 75 U. tn the present study, the material had been further fractionated by electrophoresis at pH 7.5 and 5.0 and is therefore not comparable. In the present investigation no stimulation by somatomedin A on the longitudinal bone growth was found for total doses of 14 and 70 U. This might be due to the dose levels being too low, or to some as yet. unknown factor, such as rapid inactivation or excretion in vivo or species specificity. Also, contaminating peptides in the preparation may have had a negative influence on the effect of the purified somatomedin A. Recent in viva studies with somatomedin A given to hypophysectomized rats have shown a significa.nt stimulatory influence on the sulfate incorporation into
Sohatomedin
and bone gpowth
221
skin (Fryklund et al., 1976) and costal cartilage (unpublished). NO definite effect, however, was found on the longitudinal bone growth in the present study, which might indicate a previously unr~co~n~ed specificity for the somatomedin A effect in different tissues. The present results contribute some preliminary findings to the scarce information on eventual in vivo effects of the somatomedins. Further investigations with higher dose levels and more purified preparations would be of great interest.
ACKNOWLEDGEMENTS The investigation was supported by the Faculty of Medicine, University of Lund and was carried out within a research organization sponsored by the Swedish Medical Research Council (Project 17 X-203 2).
REFERENCES Asling, C.W. and Evans, H.M. (1956) In: The Biochemistry and Physiology of Bone, Ed.: G.H. Bourne, (Academic Press, New York) pp. 671-703. Fryklund, L., Uthne, K. and Sievertsson, H. (1974) Biochem. Biophys. Res. ~ommun. 61, 957-962.
Fryklund, L., Skottner, A. and Sievertsson, H. (1976) In: Proc. 3rd Int. Congr. Growth Hormone (Excerpta Medica, Amsterdam) in press. HaU, K. (1970) Acta Endocrinol. (Kbh.) 63, 338-350. Ha&K. (1972) Acta Endocrinol. (Kbh.) suppl. 163. Hall, K., Takano. K., Fryktund, L. (1974) J. Clin. Endocrinol. 39,973-976. Hall, K., Takano., K., Fryklund, L. and Sievertsson, H. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. HaII (Academic Press, New York) pp. 19-46. Luft, R. and Hall, K. (1975) (Eds.) Somatomedins and Some Other Growth Factors. Adv. Metab. Dis., Vol. 8 (Academic Press, New York). Siovertsson, H., Ftyklund, L. Uthne, K., Hall, K. and Westermark, B. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. Hall (Academic Press, New York) pp. 47-60. Takano, K., HalI, K., Fryklund, L., Holmgren, A., Sievertsson, H. and Uthne, K, (1975) Acta Endocrinol. (Kbh.) 80, 1. Thorngren, K.-G. and Hansson, L.I. (1974a) Acta Endocrinol. (Kbh.) 75,653-668. Thorngren, K.C. and Hansson, L.I. (1974b) Acta Endocrinol. (Kbh.) 75,669-682. Thorngren, K.G. and Hansson. L.I. (1974~) Acta Endocrinol. (Kbh.) 76,35-52. Urist, M.R. (1972) In: The Biochemistry and Physiology of Bone, Vol. II. Ed.: G.H. Bourne (Academic Press, New York) pp. 155-195. Uthne, K. (1975) In: Adv. Metab. Dis., Vol. 8, Eds.: R. Luft and K. Halt. (Academic Press, New York) pp. 11.5-126.