Bone and Mineral. 177 (1992) 177-186 ~169-~09~92i$O5.00 0 1992 Elsevier Science Publishers B.V. All rights reserved.
177
BAM 00462
increases grow t osteocakin concentration in newborn
V. Coxama, lW 1. Daviccoa, P. Pastoureaub, P.D. Delmasb and .I,,$. BarleP ‘M‘t~mboiimeMinPrai, INRA Theix, St Gen&-Champanelle,France ‘insert U234. H&p&alEdouard Herriot, Lyon, Frame
(Received 23 December 1991) (Accepted 16 April 1992)
The influence of OH, IGF-I and IGF-II on plasma 1.25.(0H)sD and osteocalcin (OC) concentrations was studied in four groups of tit&:newborn lambs each. They received a single i.v. injection of either purified bovine GH (1 IU/mg 600 fig/kg body wt), synthetic human IGF-I (IO or 200 pg/kg body WS)or synthetic human IGF-II (10 @g/kgbody wt). Five controls were injected the same volume f 1 ml) of solvent. IGF-II, but not GH or IGF-I, induced a rapid (I.5 h) and sustained (up to 18 h) increase of serum osteocalcin. This effect of IGF-II on plasma OC concentration was not mediated by 1,25-(OH)zD, which increased in plasma only 18 h after IGF-II injection. These data suggest that IGF-II stimulates, the in vivo secretion of OC by osteoblasts through a mechanism independent of 1,25-(0H)zD. Further studies are necessary to eiu~date the mechanism of IGF-II action on osteocalvn synthesis and to explain the bate increase of plasma 1,25-(OH)* D after IGF-II injection.
Key words: GH; IGF-I; IGF-II; Osteocalcin; 1,25-(OH)2D, Lamb
Introduction
Insulin-like growth factors 1 (IGF-I) and 2 (IGF-II) are anabolic polypeptides which play a major role in the regulation of skeletal growth [1,2]. The presence of IGF-I and IGF-II receptors has been demonstrated on osteoblast [33 and chondrocyte [4] membranes. Trabecular and cortical bone growth is regulated by IGFs. Both IGFs are mitogenic for osteoblasts, stimulate osteoblastic synthesis of collagen [5,6] and have been isolated from bone matrix [7,8]. The IGF-11 Correspondence to: J.P. Barlet, Metabolisme Mineral, INRA Theix, F-63122 St Gen&Champanelle, France.
178 levels found in bone (about 800 pgjkg wet weight bone) are ten-fold higher than that found for IGF-I [8]. It has been shown that IGF-II is the major insulin-like growth factor produced by cartilage cells in the growth plate of immature and growing rats 191.Human IGF-II structure is thought to be identical to human skeletal growth factor (SGF) [IO]which has been isolated from human bone [l l] and extensively studied, SGF is produced by human osteoblast-like cells in culture [12], incorporated into bone matrix and might be released during osteoelastic bone resorption in a bioactive form that might influence osteoblast activity [13,14].These data suggest that IGF-II is involved in coupling bone formation to bone ~so~tion [lo] and that IGF-II might be a major regulator of bone cell metabolism [ 153. Osteocalcin (OC) is a vitamin-K- and vitamin-D-dependent protein [l&l 73 and is a specific product of the osteoblasts and odontoblasts. Serum OC is a sensitive and a specific marker of osteoblastic activity and bone fo~ation [18, 19, 201.It has been shown that serum osteocalcin is depe,rdent on GH secretion in vivo [21,22], and that IGF-I stimulates osteocalcin secretion by osteoblasts in vitro [23]. To our knowledge, the effect of IGF-II on osteocalcin secretion has never been tested. The extra~ellular concentration of inorganic phosphorus (P), and ~nsequen~y the amount of P available for cellular metabolism and growthimainly depends upon the capacity of renal tubules to reabsorb P. Conversely the tubular capacity to reabsorb P, as assessed by determining the maximal rate of P reabsorption per unit volume of glomerular filtrate, is markedly influenced by the growth rate of the organism [24,25], However, IGF-I is required for elevation of serum 1,25dihydroxyvitamin Da (1,259(OH)zDs)in response to P deprivation [26,27]. The administration of IGF-I in hypophysectomized ra.ts increases both the tubular reabsorption of P and 1,25-(OH)zDs plasma concentration. Thus IGF-I might be an important factor in the control of P metabolism during growth, and it might mediate the efTect of growth hormone (GH) on the renal handling of P and 1,25-(OH)2D~synthesis [28]. Nowadays, in France, hypotrophy is a major problem in newborn lambs [29]. Recent studies qualified newborn lamb as a good model for bone growth studies. Using the same specific radioimmunoassay for OC used in the present study, Pastoureau et al. [30] have demonstrated that OC levels can detect normal versus retarded bone growth in lambs. Furthe~ore with this animal model, a treatment with GH-releasing hormone has shown modifications of bone growth patterns by influencing dynamic histomorphometric parameters (311,Thus, in the work reported here, we have compared the effects of IGF-I and IGF-II on plasma 1,2S(OH)2Dand OC con~ntrations in normal newborn lambs. Materialsand Methods
Animals and treatment
Twenty-five 3-day-old single male lambs of the Limousine breed, spontaneously
179
born at term (146 + 1 days) were used. Each lamb was left with its mother and suckled ad libitum. They were randomly divided into five groups of five animals, The first group (birthwei~ht: 3.7 rf: 0.2 kg) received purified bovine GH (1 IU/mg; 600 pg/kg body wt; from Intervet, Angers, France). The second (birthweight: 3.9 t_ 0.3 kg) and third group (birthweight: 3.80 -t_0.4 kg) received synthetic human IGF-I (molecular weight 7646; from Ciba Geigy, Basel, Switzerland) at a dose of 10 or 200 @g/kgbody wt, respectively. The fourth group (birthweight: 3.9 f 0.5 kg) received synthetic human IGF-II (molecular weight 7471; IO pg/kg body wt; from Eli Lilly, Indianapolis, IN). The animals of the fifth group (birthweight: 3.7 f 0.1 kg), used as controls, received the same volume (1 ml) of solvent (0.9% NaCl, with 0.1% bovine serum albumin) alone, Each treatment was given as a quick bolus injection into the right jugular vein. Serial hepariuited blood samples were collected just before and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 18 and 24 h after treatment by puncture of the left jugular vein. After centrifugation plasma was frozen until analysis. Assay procedure
Plasma OC concentration was measured using a specific radioimmunoassay (RIA) for ovine OC that uses purified intact ovine OC as a standard tracer and immunogen [31]. In our conditions, the intra- and inter-assay variation was 5% and 6% respectively. Plasma IGF-I concentrations were determined by RIA after extraction [32]. In order to dissociate and separate IGF-I from its carrier protein, lamb plasma was mixed with 4 volumes of 0.5 M hydrochloric acid and incubated in stoppered glass tubes at room temperature 1331.After incubation, an ODS-silica extraction was performed (Sep-Pak Cl8 cartridges, Waters Associates, Milford, MIA). The efficiency of IGF-I extraction in ovine plasma was examined by adding synthetic human IGF-I (from Ciba Geigy, Basel, Switzerland) to 0.5-ml portions of lamb plasm,. The RIA dose-response curves of these samples paralleled those of pure IGF-I alone, but only 86 f 3% of the added IGF-I could be detected. Thus the results were corrected according to this extraction efficiency. In our experimental conditions intra- and inter-assay variations were 7% and IO%, respectively. The minimum det~~~ble amout was less than 30 p&/ml. Plasma 1,25-(0H)zD concentrations were measured using a previously described radioreceptor assay. The method is based on a thymus receptor that is specific for both 1,25-(OH)2Dz and l,25(OH)zD3. The assay involves a preliminary extraction and subsequent purification of vitamin D metabolites from plasma using CisGH ca~ridge [34].Ca and P ~on~ntrations in plasma were measured by atomic absorption spectrophotometry (Perlcin Elmer 400) and by calorimetry, respectively. Results are expressed as means + SEM. The statistical signi~can~e of differences observed between groups was calculated using Mann-Whitney U-test. Oneway analysis of variance was also used to compare values within one QOUP of animals.
180
100_
80.
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gis _ 5 40. 20. 01
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TIME
Fig. I. Plasma IGF concentrationsmeasuredin lambs injected with GH (black triangle) IGF-I (200 &kg body wt; black squares) or IGF-II (black circles). Results are expressed as means f SEM. “P,O.OS, “P30.01 (compa~son with control lambs, dotted line, open circIes). For clarity. Iambs given IO@gIGFI/kg body wt (which were never different from controls) are not shown in the figure.
IWO
1t
900.
800. : 700_ g!? U 600_ soo_ 4ooJ
I..~-...0 Q5
...1 1.5
4
18
24 Hours
TIME Fig. 2. Plasma osteocalcin {GC) concentrations measured in iambs injected with GH {black triangles),
IGF-I (200 @kg body wt; black squares) or IGF-II (black circles). Results are expressed as means &SEM.’ P,O.OS, **P20.01 (comparison with control lambs, dotted line, open circles). For clarity, lambs given 10 pg IGF-I/kg body wt {which were never different from controls) are not shown in the figure.
181 130
120
110
70.
60
50
40.
L--...I.-.
0
1
1.5
-
4
18
24
Hours TIME
Fig. 3. Plasma 1,25-(OH)zD concentrations measured in lambs injected with GH (black triangles), IGF-I (200 pg/kg body wt black squares) or IGF-II (black circles). Results are expressed as means +SEM. *P>O.O5, “PaO.01 (comparison with control lambs, dotted line, open circles). For clarity, lambs given IO pg IGF-I/kg body wt (which were never different from controls) are not shown in the Figure.
Results
The intravenous injection of GH induced a significant increase in plasma IGF-I concentration at 30 min. The effect disappeared 30 min later. Plasma IGF-I concentrations were significantly increased from 30 min until 18 h after injection in lambs given 200 fig IGF-I/body wt. In those receiving only 10 ,ug IGF/kg body wt, plasma IGF-I concentrations were never different from those measured in controls (Fig. 1). Neither GH nor IGF-I (10 or 200 pg/kg body wt) had any significant effect on plasma OC concentrations (Fig. 2). Eighteen hours after injection, GH and IGF-II induced an increase in plasma 1,25-(OH)7_Dconcentrations from 61 + 4 pg/ml to 86 f 3 pg/ml (P < 0.05) and from 65 + 5 pg/ml to 122 + 11 pg/ml (P < O.Ol), respectively (Fig. 3). IGF-II injection induced a significant increase in plasma OC and 1,2S-(0H)zD concentrations. The rise in plasma OC started 90 min after injection while that in plasma 1,25-(OH)*D was effective ouly at 18 h. (Figs. 2 and 3). No significant relationship could be demonstrated between plasma OC and 1,25-(0H)iD con-
182 12.5
C.
0
*
1
f
1
1
2
-..,-
.,L.,
4
18
24 Hours
TlWlE
Fig. 4 Plasma calcium (Ca) and inorganic phosphorus (P) concentrations measured in lambs injected with GH (black triangles), IGF-I (200 &kg body wt; black squares) or IGF-II (black circles). Results are expressed as means f SEM. No significant difference was observed between groups.
centrations. No significant variation in plasma Ca (mean value 11.2 + 0.1 mgjdl) and P (mean value 6.1 f 0.1 mg/dl) concentration was observed in any group of lambs (Fig. 4).
Our results (Fig. I) bones previous studies demonstrating that in newborn mammals GH is able to stimulate hepatic IGF-I production and to increase plasma IGF-I concentration (Fig. 1; [35]). It has also been previously demonstrated that GH can stimulate renal la-hydroxylase activity and 1,25-(OH)zDs production [36],an effect possibly mediated through an increase in plasma IGF-I concentration [26, 27, 281, and that IGF-I was able to stimulate renal 1,25(OH)zD3 synthesis by regulating the renal handling of P in hypophysectomized rats [28]. In the newborn lambs used in this expe~m~nt, only GH and IGF-II t~atments had a signi~cant effect upon plasma 1,25-(0H)zD concentrations (Fig. 3). In these animals, GH treatment increased plasma 1,25-(OH)zD concentration up to 86 f 3 pgjml at 18 h, However, in our experimental conditions, these effCkcts of GH on plasma IGF-I and 1,25-(OH)zDconcentrations were slight and transient (Figs. 1 and 3). The lack of effect of GH upon plasma OC levels might be explained in two ways: l Although endogenoas plasma GH concentrations measured in newborn
183
lambs are lower than fetal levels [373, they are however higher than those measured in adult sheep 137,383.In the human species, exogenous GI-I has been demonstrated to increase plasma OC concentrations only when endogenous GI-I was low: Turner’s syndrome [39], postmenopausal women [40] and GII-deficient adults [41] or children [21]. bGH might be without effect upon bone growth in newborn lambs. It has been shown to increase daily weight gain in hy~phys~ctomized 2-month-old lambs [42], but it has no significant effect on. body weight or daily weight gain in normal suckling lambs during the first month of postnatal life [42,43]. Ovine IGFs are very similar to those of human and bovine in structure and activity [44]. Thus minor responses observed in plasma OC and 1,25-(OH)~~ concentrations following IGF-I treatment (Figs. 2 and 3), probably did not result from the use of heterologous (human) peptides in the ovine species. In contrast, plasma OC was significantly increased following IGF-II injection (Figs, 2 and 3), which had no significant effect on plasma IGF-I concentration (Fig, I). Plasma OC concentrations measured in our 3-day-old lambs (607 f 83 ng~ml) are in good agr~ment with those already reported in newborn lambs 1301.In sheep, like in other mammals, plasma OC concentration is strongly correlated to OC osteoblastic production and to bone growth [46]. The physiological role of OC remains unknown [47]. IIuman IGF-II, the amino acid sequence of which is identical to that of human skeletal growth factor [lo], can have both autocrine and paracrine functions on cells of the osteoblast line. It has been interpreted to be a putative coupling factor present in bone matrix and released by bone resorption to increase osteoblast, number and thereby increase the bone formation rate [I I]. To our knowledge, our results represent the first evidence that a single injection of IGF-II is able to increase (by a still unknown mechanism) plasma OC concentrations in newborn lambs without changing plasma IGF-I. Because 1,25-(OH)& is known to stimulate OC gene transcription [48] and to increase OC production in vitro [49] and in vivo [50], the effect of IGF-II on pfasma OC concentration might be mediated through an increase in plasma 1,25(OH)zI3. This was not the case since such an increase was observed only after the rise of plasma OC concentration (Figs. 2 and 3). Because the increase of serum osteocalcin occurred rapidly, within 90 min after IGF-II injection, it is likely to reflect a direct effect of IGF-II on osteocalcin secretion at the cell level rather than an effect of IGF-II on bone fo~ation. Although the increase in plasma OC concentration observed following IGF-II injection might result from a decrease in OC catabolism, this was probably not the case since plasma OC concentration appears as a very good index of osteoblast function in the ovine species due to a constant OC metabolic clearance rate in these animals [Sl]. It has been demonstrated that GH and 1,25-(OH)z~ may modulate the metabolism of human osteoblasts by regulating local production of IGF-I [23,53]. In vitro studies using fetal rat bones have also shown that IGF-II binds primarily to a large number of high-affinity IGF-II receptors, but may ah occupy IGF-I binding sites, and IGF-II easily displaces ‘%IGF-I by comparison to the ability of IGF-I to displace 1251-IGF-II1543.In our experiment, on a
184
was more potent than IGF-I to increase plasma OC concentration. In the ovine species, it has been demonstrated that plasma IGF-II concentrations decrease at birth while plasma IGF-I concentrations increase [SS]. In fact basal plasma IGF-I concentrations were as high as 20 nM (about 150 ng/ ml) in our animals (Fig. 1): this might also explain why exogenous IGF-I was ine~~tive. Finally, in suckling newborn mammals (and also possibly in fetuses) whose phosphataemia is high (> 6 mg/dl) IGF-I appears without effect on plasma OC and 1,25-(OH)zD concentrations (Figs. 2 and 3); in adult rats the stimulatory effect of IGF-I on plasma 1,25(OH)~D~concentration was more intense after restriction of dietary phosphorus, inducing plasma phosphorus concentration around 3 mg/dl[27]. On the other hand, the recent demonstration that hone cells produce multiple IGF-binding proteins suggests that these binding proteins may ultimately dete~in~ the manifestation of IGF-I or IGF-II action fS2]. In conclusion, in suckling newborn lambs the intravenous injection of a low dose of IGF-II (about 1 nmol/kg body wt) increased plasma OC and 1,25(OH)zD concentrations. In these animals a twenty times higher dose of IGF-I had no si~ni~~ant ef%ct on these parameter. molar basis,
IGF-II
The lambs used in this study were kindly supplied by the Laboratoire de Production Ovine. IGF-I and IGF-II were generous gift from Dr. J. Nuesch (Ciba Geigy, Base&Switzerland) and Dr. T.J. Jeatran (Lilly Research Laboratories, Indianapolis, USA), respectively. References I Froesch ER, SchmidC, SchwanderJ, Zapf. J Actions of insulin-likegrowth factors.Annu Rev Physiol 198$47:443-4677. 2 SpencerEM, Liu CC, Si ECC, HowardGA. fn vivo actions of insulin-likegrowth factor-1(fGF-f) on bone fo~ation and caption in rats. Bone 1~1;12:21-26. 3 Siootweg MC, HoogerbruggeCM, De PoorterTL, Duursma SA, Van Buul-OffersSC. The presence of classical insulin-likegrowth factor (IOF) type=1and II receptorson mouse osteoblasts autocrine/ paracrinegrowth effects of IGFs? J Endocrinol 1990;125:271-277. 4 Jansen J. Van Buul-OffersSC, Hoogerb~~e CM, De Poorter TJ, Corvol MT, Van Den BrandeJL. Characterizationof specific insulin-like growth factor (lGF)-I and II receptors on cultured rabbit articularchondrocyte membranes.J Endocrinol 1989;120:245-249. 5 Canalis EM. Effect of insulin-like growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 1980;66:709-719. 6 ~hmid C, Steiner R, Froesch ER. Insulin-likegrowth factors stimulatesynthesis of nucleic acids and glycogen in cultured calvaria cells, Calcif Tissue Int 1983;35:578-585. 7 Canalis E, McCarthyT, Centrclla M. Isolation and characterizationof insulin-like growth factor I (somatomedin C) from cultures of fetal rat calvariae. Endocrinology 1988;122:22-27. 8 Frolik CA, Ellis LF, Williams DC. Isolation and chara~te~~tion of insulin-like growth factor II from human bone. Biochem Biophys Res C;>mmun 1988;151:1~11-1018.
185 9 Shinar DM, Rodan GA, Weinreb M. Differential expression of IGF-I and IIGF-II in growing rat bone. J Bone Miner Res 1991; 6 (Suppl I): S251. 10 Mohan S, Jennings JC, Linkhart TA, Baylink DJ. Primary structure of human skeletal growth factor: homology with human insulin-like growth factor-Ii. Biochim Biophys Acta 1988;966:44.-55. 11 Farley JR, Baylink DJ. Purification of a skeletal growth factor from human bone. Biochemistry 1982;21:3502-3507. 12 Wergedal JE, Mohan S, Taylor AK, Baylink DJ. Skeletal growth factor is produced by human osteoblast-like cells in culture. Biochim Biophys Acta 1986;88~163-170. 13 Mohan S, Linkhart T, Farley J, Baylink D. Bone derived factors active on bone cells. Calcif Tissue ]nt 1984;36:5139-S145. 14 Farley JR, Tarbaux N, Murphy LA, Masuda T, Baylink DJ. In vitro evidence that bone formation may be coupled to resorption by release of mitogen(s) from resorbing bone. Metabolism 1987;36:314321. 15 Baylink DJ, Mohan S. Insulin-like growth factors and their biuding proteins, J Bone Miner Res 1991;6 (Suppl I):XV. 16 Hauschka PV. Lian BJ, Gallop PM. Direct identification of the calcium binding amino acid gamma car~xy8lutamate in mineralized tissue, Proc Nat1 Acad Sci USA 1975;72:392S-3929. 17 Price PA, WilIia~nsonMK, ~athinger JW. Origin of the v*itaminK-dependent bone protein found in plasma and its clearance by kidney and bone. J Biol Chem 1981;256:12760-I2766. 18 Price PA, Parthemore JO, Deftos LJ. New biochemical marker for bone metabolism. J Clin Invest 1980;66:878-883. 19 Delmas PD. Wahner HW, Mann KG, Riggs BL. Assessment of bone turnover in postmenopausal osteoporosis by m~sureme~t of serum bone gla-protein. J Lab Clin Med 1983;10~47~76. 20 Deimas PD, Derniaux B, Malaval L, Chapuy MC, Edouard C, Meunier PJ. Serum bone-gla protein (osteocalcin) in primary hyperparathyroidism and in malignant hypercalcemia. Comparison with bone histomorphometry. J Clin Invest 1986;77:985-991. 21 Delmas PD, Chatelain P, Bonne G. Serum bone Gla-protein in growth hormone deficient children. J Bone Miner Res 1986:1:333-338. 22 Johansen JS. Giwercman A, Hartwell D, Nielsen CT, Price PA, Christiansen C, Skakkebaek NE. Serum bone gla-protein as a marker of bone growth in children and adolescents: correlation with age, height, serum insulin-like growth factor I, and serum testosterone. J Clin Endocrinol Metab i988;67:273-278. 23 Chenu C, Valentin-Opran A, Chavassieu~ P, Saez S, Meunier PJ, Delmas PD. Insulin-like growth factor I hormonal regulation by growth hormone and by 1,25-(OH)zDj and activity on human osteoblast-like ceils in short-term cultures. Bone 1990;11:81-86. 24 Corvilain J, Abramor M. Growth and renal control of plasma phosphate. J Clin Endocrinol Metab 1972;34:452-459. 25 Caversazio 9, Bonjour JP, Fteish H. Tubular handling of Pi in young growing and adult rats. Am J Physiol 1982;242:F7QS-F710 26 Gray R. Evidence that somatomedins mediate the effect of hypophosphatemia to increase serum 1,25dihydroxyvitamin DJ levels in rats. Endocrinology 1987; I21 304-512. 27 Hallorat: BP, Spencer EM. Dietary phosphorus and 1.25.dihydroxyvitamin D metabolism: influence of insulin-like growth factor I. End~rinoIogy 1988;123:1225-1229. 28 Caversazio J, Montessut C, Bonjour JP. Stimulatory effect of insulin-like growth factor-1 on renal Pi transport and plasma 1,2$dihydroxyvitamin Ds. Endocrinology 1990;127:4S3-459. 29 Vihette Y, Theriez M. Influence du poids ri la naissance sur les performances d”agneaux de boucherie. Ann Zootech 1981;30:151-i81. 30 Pastoureau P, Merle B, Delmas PD. Specific radioimmunoassay for ovine bone gIa-Protein (osteocalcin). Acta Endocrinol 1988;119;152--160. 31 Pastoureau P, Charrier J, Blanchard M, Boivin G, Dulor JP. Theriez M, Barenton B. Effects of a chronic GRF treatment on lambs having low or I;ormal birthweights. Domestic Anim Endocrinol 1989;6:32l-329. 32 Coxam V, Davitxo MJ, Dardillat C, Gpmeer F, Barlet JP. Systemic bone growth factors concentrations in calves during the perinatal period. J Develop Physiol 1988;1~423~3~.
186 33 Underwood LE, D’Ercole VA, Copeland KC, Van Wyk JJ, Hurley T, Handwerger S. Deve!oPmentof an heterologous radioimmunoassay for somatomedin C in sheep blood. J Endocrinol 1982;93:3!-39. 34 Hollis BW, Assay of circulating 1,25dihydroxyvitamin D involving a novel single cartridge extraction and purification procedure. Clin Chem 1986;32:2060-2063. 35 coxam V, Davicco MJ, Opmeer F, Beckers JF, Durand D, Bauchart D, Barlet JP. Endocrine relation ofhepatic somatom~in C (IGF-f) production in young calves. In: Jones CT, ed. Fetal and neonatal development. New York: Perinatology Press 1988;496-499. 36 SPanos E, Barret D, Macintyre I, Pike JW, Salilian EC, Haussler MR. Effect of growth hormone on vitamin D metabolism. Nature 1978;273:246-247. 37 Gluckman PD. Mueller PL, Kaplan SL, Rudolph AM, Grumbach MM. Hormone ontogeny in the ovine fetus, I, Ci~ulating growth ho~one in mid and late gestation. Endocrinology 1979;104:!62168. 38 Bessett JM, Thorburn GD, Wallace ALC. The plasma growth hormone concentration in the fetal lamb, J Endocrinol 1970;48:25!-263. 39 Bergmann P, Valsamis 3, Van Perborgh J, De Schepper J, Van Vliet G. Comparative studies of the changes in insulin-like growth factor-i, procollagen-III N-terminal extension peptide, bone glaprotein, and bone mineral content in children with Turner’s syndrome treated with r~ombinant growth hormone. J Chin ~t~docrinol Metab 19~;71:1461-1~7. 40 Franchimont P, Urbain-Choffrey D, Lambelin P, Fontaine MA, Frangin G, Reginster JY. Effects of repetitive administration of growth hormone-releasing hormone on growth hormone secretion, insulin-like growth factor-1 and bone metabolism in postmenopausal women. Acta Endocrinol 1989;!20:121-128. 41 Johansen JS, Pedersen SA, Jorgensen JOL, Reis BJ, Ch~stiansen C, Christian~n JS, Skakke~eck NE. Effects of growth hormone (GH) on plasma bone gla protein in GH-deficient adults. J Clin Endocrinol Metab 1990;70:916-919. 42 Vezinhet A, Dauzier L. Influence de traitements ti, l’hormone somatotrope bovine sur la croissance pond&ale d’agneaux normaux ou hypoptrysectomids. Ann Biol Anim Biochim Biophys 1970;10:5--13. 43 Koppel J, Kuchar S, Rynikova A, Mazes S, Noskovic P, Boda K. Effects of bovine growth hormone in suckling lambs. Exp Clin ~nd~rinol l988;91:223-226. 44 Francis GL, McNeil KA, Wallace JC, Ballard FJ, Owens PC. Sheep insulin-like growth factors I and II: sequences, activities and assays. Endocrinology 1989;124:1173-I 183. 45 Farrugia W, Fortune CL. Heath 3. Caple IW, Wark JD. Osteocalcin as an index of osteoblast function during and alter ovine pregnancy, Endocrinology 1989;125:1705-I 7 10. 46 Pastou~au P, Meunier PJ, Delmas P, Serum oste~alcin (bone gla-protein), an index of bone growth in lambs. ~orn~~son with age-related histomo~hometric changes. Bone 1991;!~143-149. 47 Baylink JD, Taylor AK, Johnston P, Arnaud S, Lueken S, Linkhart SG. Update on osteocalcin assays, Calcif Tissue lnt 1991;48:(Suppl):A4(Abstract). 48 Pan LC, Price PA. The effect of transcriptional inhibitors on the bone-carboxyglutamic acid and protein response to !.2S-dihydroxyvitamin D3 in osteosarcoma cells. J Biol Chem 1984;259:58445847. 49 Skjodt H, Gallagher JA, Bersford JN, Couch M, Poser JW. Russel RGG. Vitamin D metabolites regulate osteoca!cin synthesis and proliferation of human bone cells in vitro. J Endocrinol 1985:105:391-396. 50 Price PA, Bauko! SA. !,25-dihydroxyvitamin D3 increases serum levels of the vitamin K-dependent bone protein. Biochem Biophys Res Commun 1981;9~928-935. 51 Melick RA, Farrugia W, Heaton CL, Quelch KJ, Scoggins BA, Wark JD. The metabolic clearance rate of osteocalcin in sheep. Calcif Tissue Int 1988;42:185-190. $2 Mohan S, Baylink D. Isolation and characterization of insulinlike growth factor binding proteins (IGFBPs) produced by human bone cells (HBC) in vitro. J Bone Mit Res 1991;6 (Suppl l):A233. 53 canalis E, Lian JB. Effects of bone associated growth factors on DNA, collagen and osteoe&in synthesis in cultured fetal rat calvariae. Bone 1988;9:243-248. 34 Cent&la M, MC Carthy TL, Canalis E. Receptors for insulinlike growth factors-I and -II in oskoblast-enriched cultures from fetal rat bone. Endocrinology 1990;!26:39-44. 58 Gluckmau PD, Butler JH. Parturition-related changes in insulin-like growth factor I and, II in the perinata! lamb. 3 Endocrinol 1983;99:223-232.
177
BAM 00462
increases grow t osteocakin concentration in newborn
V. Coxama, lW 1. Daviccoa, P. Pastoureaub, P.D. Delmasb and .I,,$. BarleP ‘M‘t~mboiimeMinPrai, INRA Theix, St Gen&-Champanelle,France ‘insert U234. H&p&alEdouard Herriot, Lyon, Frame
(Received 23 December 1991) (Accepted 16 April 1992)
The influence of OH, IGF-I and IGF-II on plasma 1.25.(0H)sD and osteocalcin (OC) concentrations was studied in four groups of tit&:newborn lambs each. They received a single i.v. injection of either purified bovine GH (1 IU/mg 600 fig/kg body wt), synthetic human IGF-I (IO or 200 pg/kg body WS)or synthetic human IGF-II (10 @g/kgbody wt). Five controls were injected the same volume f 1 ml) of solvent. IGF-II, but not GH or IGF-I, induced a rapid (I.5 h) and sustained (up to 18 h) increase of serum osteocalcin. This effect of IGF-II on plasma OC concentration was not mediated by 1,25-(OH)zD, which increased in plasma only 18 h after IGF-II injection. These data suggest that IGF-II stimulates, the in vivo secretion of OC by osteoblasts through a mechanism independent of 1,25-(0H)zD. Further studies are necessary to eiu~date the mechanism of IGF-II action on osteocalvn synthesis and to explain the bate increase of plasma 1,25-(OH)* D after IGF-II injection.
Key words: GH; IGF-I; IGF-II; Osteocalcin; 1,25-(OH)2D, Lamb
Introduction
Insulin-like growth factors 1 (IGF-I) and 2 (IGF-II) are anabolic polypeptides which play a major role in the regulation of skeletal growth [1,2]. The presence of IGF-I and IGF-II receptors has been demonstrated on osteoblast [33 and chondrocyte [4] membranes. Trabecular and cortical bone growth is regulated by IGFs. Both IGFs are mitogenic for osteoblasts, stimulate osteoblastic synthesis of collagen [5,6] and have been isolated from bone matrix [7,8]. The IGF-11 Correspondence to: J.P. Barlet, Metabolisme Mineral, INRA Theix, F-63122 St Gen&Champanelle, France.
178 levels found in bone (about 800 pgjkg wet weight bone) are ten-fold higher than that found for IGF-I [8]. It has been shown that IGF-II is the major insulin-like growth factor produced by cartilage cells in the growth plate of immature and growing rats 191.Human IGF-II structure is thought to be identical to human skeletal growth factor (SGF) [IO]which has been isolated from human bone [l l] and extensively studied, SGF is produced by human osteoblast-like cells in culture [12], incorporated into bone matrix and might be released during osteoelastic bone resorption in a bioactive form that might influence osteoblast activity [13,14].These data suggest that IGF-II is involved in coupling bone formation to bone ~so~tion [lo] and that IGF-II might be a major regulator of bone cell metabolism [ 153. Osteocalcin (OC) is a vitamin-K- and vitamin-D-dependent protein [l&l 73 and is a specific product of the osteoblasts and odontoblasts. Serum OC is a sensitive and a specific marker of osteoblastic activity and bone fo~ation [18, 19, 201.It has been shown that serum osteocalcin is depe,rdent on GH secretion in vivo [21,22], and that IGF-I stimulates osteocalcin secretion by osteoblasts in vitro [23]. To our knowledge, the effect of IGF-II on osteocalcin secretion has never been tested. The extra~ellular concentration of inorganic phosphorus (P), and ~nsequen~y the amount of P available for cellular metabolism and growthimainly depends upon the capacity of renal tubules to reabsorb P. Conversely the tubular capacity to reabsorb P, as assessed by determining the maximal rate of P reabsorption per unit volume of glomerular filtrate, is markedly influenced by the growth rate of the organism [24,25], However, IGF-I is required for elevation of serum 1,25dihydroxyvitamin Da (1,259(OH)zDs)in response to P deprivation [26,27]. The administration of IGF-I in hypophysectomized ra.ts increases both the tubular reabsorption of P and 1,25-(OH)zDs plasma concentration. Thus IGF-I might be an important factor in the control of P metabolism during growth, and it might mediate the efTect of growth hormone (GH) on the renal handling of P and 1,25-(OH)2D~synthesis [28]. Nowadays, in France, hypotrophy is a major problem in newborn lambs [29]. Recent studies qualified newborn lamb as a good model for bone growth studies. Using the same specific radioimmunoassay for OC used in the present study, Pastoureau et al. [30] have demonstrated that OC levels can detect normal versus retarded bone growth in lambs. Furthe~ore with this animal model, a treatment with GH-releasing hormone has shown modifications of bone growth patterns by influencing dynamic histomorphometric parameters (311,Thus, in the work reported here, we have compared the effects of IGF-I and IGF-II on plasma 1,2S(OH)2Dand OC con~ntrations in normal newborn lambs. Materialsand Methods
Animals and treatment
Twenty-five 3-day-old single male lambs of the Limousine breed, spontaneously
179
born at term (146 + 1 days) were used. Each lamb was left with its mother and suckled ad libitum. They were randomly divided into five groups of five animals, The first group (birthwei~ht: 3.7 rf: 0.2 kg) received purified bovine GH (1 IU/mg; 600 pg/kg body wt; from Intervet, Angers, France). The second (birthweight: 3.9 t_ 0.3 kg) and third group (birthweight: 3.80 -t_0.4 kg) received synthetic human IGF-I (molecular weight 7646; from Ciba Geigy, Basel, Switzerland) at a dose of 10 or 200 @g/kgbody wt, respectively. The fourth group (birthweight: 3.9 f 0.5 kg) received synthetic human IGF-II (molecular weight 7471; IO pg/kg body wt; from Eli Lilly, Indianapolis, IN). The animals of the fifth group (birthweight: 3.7 f 0.1 kg), used as controls, received the same volume (1 ml) of solvent (0.9% NaCl, with 0.1% bovine serum albumin) alone, Each treatment was given as a quick bolus injection into the right jugular vein. Serial hepariuited blood samples were collected just before and 0.25, 0.5, 0.75, 1, 1.5, 2, 4, 18 and 24 h after treatment by puncture of the left jugular vein. After centrifugation plasma was frozen until analysis. Assay procedure
Plasma OC concentration was measured using a specific radioimmunoassay (RIA) for ovine OC that uses purified intact ovine OC as a standard tracer and immunogen [31]. In our conditions, the intra- and inter-assay variation was 5% and 6% respectively. Plasma IGF-I concentrations were determined by RIA after extraction [32]. In order to dissociate and separate IGF-I from its carrier protein, lamb plasma was mixed with 4 volumes of 0.5 M hydrochloric acid and incubated in stoppered glass tubes at room temperature 1331.After incubation, an ODS-silica extraction was performed (Sep-Pak Cl8 cartridges, Waters Associates, Milford, MIA). The efficiency of IGF-I extraction in ovine plasma was examined by adding synthetic human IGF-I (from Ciba Geigy, Basel, Switzerland) to 0.5-ml portions of lamb plasm,. The RIA dose-response curves of these samples paralleled those of pure IGF-I alone, but only 86 f 3% of the added IGF-I could be detected. Thus the results were corrected according to this extraction efficiency. In our experimental conditions intra- and inter-assay variations were 7% and IO%, respectively. The minimum det~~~ble amout was less than 30 p&/ml. Plasma 1,25-(0H)zD concentrations were measured using a previously described radioreceptor assay. The method is based on a thymus receptor that is specific for both 1,25-(OH)2Dz and l,25(OH)zD3. The assay involves a preliminary extraction and subsequent purification of vitamin D metabolites from plasma using CisGH ca~ridge [34].Ca and P ~on~ntrations in plasma were measured by atomic absorption spectrophotometry (Perlcin Elmer 400) and by calorimetry, respectively. Results are expressed as means + SEM. The statistical signi~can~e of differences observed between groups was calculated using Mann-Whitney U-test. Oneway analysis of variance was also used to compare values within one QOUP of animals.
180
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Fig. I. Plasma IGF concentrationsmeasuredin lambs injected with GH (black triangle) IGF-I (200 &kg body wt; black squares) or IGF-II (black circles). Results are expressed as means f SEM. “P,O.OS, “P30.01 (compa~son with control lambs, dotted line, open circIes). For clarity. Iambs given IO@gIGFI/kg body wt (which were never different from controls) are not shown in the figure.
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TIME Fig. 2. Plasma osteocalcin {GC) concentrations measured in iambs injected with GH {black triangles),
IGF-I (200 @kg body wt; black squares) or IGF-II (black circles). Results are expressed as means &SEM.’ P,O.OS, **P20.01 (comparison with control lambs, dotted line, open circles). For clarity, lambs given 10 pg IGF-I/kg body wt {which were never different from controls) are not shown in the figure.
181 130
120
110
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60
50
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Fig. 3. Plasma 1,25-(OH)zD concentrations measured in lambs injected with GH (black triangles), IGF-I (200 pg/kg body wt black squares) or IGF-II (black circles). Results are expressed as means +SEM. *P>O.O5, “PaO.01 (comparison with control lambs, dotted line, open circles). For clarity, lambs given IO pg IGF-I/kg body wt (which were never different from controls) are not shown in the Figure.
Results
The intravenous injection of GH induced a significant increase in plasma IGF-I concentration at 30 min. The effect disappeared 30 min later. Plasma IGF-I concentrations were significantly increased from 30 min until 18 h after injection in lambs given 200 fig IGF-I/body wt. In those receiving only 10 ,ug IGF/kg body wt, plasma IGF-I concentrations were never different from those measured in controls (Fig. 1). Neither GH nor IGF-I (10 or 200 pg/kg body wt) had any significant effect on plasma OC concentrations (Fig. 2). Eighteen hours after injection, GH and IGF-II induced an increase in plasma 1,25-(OH)7_Dconcentrations from 61 + 4 pg/ml to 86 f 3 pg/ml (P < 0.05) and from 65 + 5 pg/ml to 122 + 11 pg/ml (P < O.Ol), respectively (Fig. 3). IGF-II injection induced a significant increase in plasma OC and 1,2S-(0H)zD concentrations. The rise in plasma OC started 90 min after injection while that in plasma 1,25-(OH)*D was effective ouly at 18 h. (Figs. 2 and 3). No significant relationship could be demonstrated between plasma OC and 1,25-(0H)iD con-
182 12.5
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1
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.,L.,
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Fig. 4 Plasma calcium (Ca) and inorganic phosphorus (P) concentrations measured in lambs injected with GH (black triangles), IGF-I (200 &kg body wt; black squares) or IGF-II (black circles). Results are expressed as means f SEM. No significant difference was observed between groups.
centrations. No significant variation in plasma Ca (mean value 11.2 + 0.1 mgjdl) and P (mean value 6.1 f 0.1 mg/dl) concentration was observed in any group of lambs (Fig. 4).
Our results (Fig. I) bones previous studies demonstrating that in newborn mammals GH is able to stimulate hepatic IGF-I production and to increase plasma IGF-I concentration (Fig. 1; [35]). It has also been previously demonstrated that GH can stimulate renal la-hydroxylase activity and 1,25-(OH)zDs production [36],an effect possibly mediated through an increase in plasma IGF-I concentration [26, 27, 281, and that IGF-I was able to stimulate renal 1,25(OH)zD3 synthesis by regulating the renal handling of P in hypophysectomized rats [28]. In the newborn lambs used in this expe~m~nt, only GH and IGF-II t~atments had a signi~cant effect upon plasma 1,25-(0H)zD concentrations (Fig. 3). In these animals, GH treatment increased plasma 1,25-(OH)zD concentration up to 86 f 3 pgjml at 18 h, However, in our experimental conditions, these effCkcts of GH on plasma IGF-I and 1,25-(OH)zDconcentrations were slight and transient (Figs. 1 and 3). The lack of effect of GH upon plasma OC levels might be explained in two ways: l Although endogenoas plasma GH concentrations measured in newborn
183
lambs are lower than fetal levels [373, they are however higher than those measured in adult sheep 137,383.In the human species, exogenous GI-I has been demonstrated to increase plasma OC concentrations only when endogenous GI-I was low: Turner’s syndrome [39], postmenopausal women [40] and GII-deficient adults [41] or children [21]. bGH might be without effect upon bone growth in newborn lambs. It has been shown to increase daily weight gain in hy~phys~ctomized 2-month-old lambs [42], but it has no significant effect on. body weight or daily weight gain in normal suckling lambs during the first month of postnatal life [42,43]. Ovine IGFs are very similar to those of human and bovine in structure and activity [44]. Thus minor responses observed in plasma OC and 1,25-(OH)~~ concentrations following IGF-I treatment (Figs. 2 and 3), probably did not result from the use of heterologous (human) peptides in the ovine species. In contrast, plasma OC was significantly increased following IGF-II injection (Figs, 2 and 3), which had no significant effect on plasma IGF-I concentration (Fig, I). Plasma OC concentrations measured in our 3-day-old lambs (607 f 83 ng~ml) are in good agr~ment with those already reported in newborn lambs 1301.In sheep, like in other mammals, plasma OC concentration is strongly correlated to OC osteoblastic production and to bone growth [46]. The physiological role of OC remains unknown [47]. IIuman IGF-II, the amino acid sequence of which is identical to that of human skeletal growth factor [lo], can have both autocrine and paracrine functions on cells of the osteoblast line. It has been interpreted to be a putative coupling factor present in bone matrix and released by bone resorption to increase osteoblast, number and thereby increase the bone formation rate [I I]. To our knowledge, our results represent the first evidence that a single injection of IGF-II is able to increase (by a still unknown mechanism) plasma OC concentrations in newborn lambs without changing plasma IGF-I. Because 1,25-(OH)& is known to stimulate OC gene transcription [48] and to increase OC production in vitro [49] and in vivo [50], the effect of IGF-II on pfasma OC concentration might be mediated through an increase in plasma 1,25(OH)zI3. This was not the case since such an increase was observed only after the rise of plasma OC concentration (Figs. 2 and 3). Because the increase of serum osteocalcin occurred rapidly, within 90 min after IGF-II injection, it is likely to reflect a direct effect of IGF-II on osteocalcin secretion at the cell level rather than an effect of IGF-II on bone fo~ation. Although the increase in plasma OC concentration observed following IGF-II injection might result from a decrease in OC catabolism, this was probably not the case since plasma OC concentration appears as a very good index of osteoblast function in the ovine species due to a constant OC metabolic clearance rate in these animals [Sl]. It has been demonstrated that GH and 1,25-(OH)z~ may modulate the metabolism of human osteoblasts by regulating local production of IGF-I [23,53]. In vitro studies using fetal rat bones have also shown that IGF-II binds primarily to a large number of high-affinity IGF-II receptors, but may ah occupy IGF-I binding sites, and IGF-II easily displaces ‘%IGF-I by comparison to the ability of IGF-I to displace 1251-IGF-II1543.In our experiment, on a
184
was more potent than IGF-I to increase plasma OC concentration. In the ovine species, it has been demonstrated that plasma IGF-II concentrations decrease at birth while plasma IGF-I concentrations increase [SS]. In fact basal plasma IGF-I concentrations were as high as 20 nM (about 150 ng/ ml) in our animals (Fig. 1): this might also explain why exogenous IGF-I was ine~~tive. Finally, in suckling newborn mammals (and also possibly in fetuses) whose phosphataemia is high (> 6 mg/dl) IGF-I appears without effect on plasma OC and 1,25-(OH)zD concentrations (Figs. 2 and 3); in adult rats the stimulatory effect of IGF-I on plasma 1,25(OH)~D~concentration was more intense after restriction of dietary phosphorus, inducing plasma phosphorus concentration around 3 mg/dl[27]. On the other hand, the recent demonstration that hone cells produce multiple IGF-binding proteins suggests that these binding proteins may ultimately dete~in~ the manifestation of IGF-I or IGF-II action fS2]. In conclusion, in suckling newborn lambs the intravenous injection of a low dose of IGF-II (about 1 nmol/kg body wt) increased plasma OC and 1,25(OH)zD concentrations. In these animals a twenty times higher dose of IGF-I had no si~ni~~ant ef%ct on these parameter. molar basis,
IGF-II
The lambs used in this study were kindly supplied by the Laboratoire de Production Ovine. IGF-I and IGF-II were generous gift from Dr. J. Nuesch (Ciba Geigy, Base&Switzerland) and Dr. T.J. Jeatran (Lilly Research Laboratories, Indianapolis, USA), respectively. References I Froesch ER, SchmidC, SchwanderJ, Zapf. J Actions of insulin-likegrowth factors.Annu Rev Physiol 198$47:443-4677. 2 SpencerEM, Liu CC, Si ECC, HowardGA. fn vivo actions of insulin-likegrowth factor-1(fGF-f) on bone fo~ation and caption in rats. Bone 1~1;12:21-26. 3 Siootweg MC, HoogerbruggeCM, De PoorterTL, Duursma SA, Van Buul-OffersSC. The presence of classical insulin-likegrowth factor (IOF) type=1and II receptorson mouse osteoblasts autocrine/ paracrinegrowth effects of IGFs? J Endocrinol 1990;125:271-277. 4 Jansen J. Van Buul-OffersSC, Hoogerb~~e CM, De Poorter TJ, Corvol MT, Van Den BrandeJL. Characterizationof specific insulin-like growth factor (lGF)-I and II receptors on cultured rabbit articularchondrocyte membranes.J Endocrinol 1989;120:245-249. 5 Canalis EM. Effect of insulin-like growth factor I on DNA and protein synthesis in cultured rat calvaria. J Clin Invest 1980;66:709-719. 6 ~hmid C, Steiner R, Froesch ER. Insulin-likegrowth factors stimulatesynthesis of nucleic acids and glycogen in cultured calvaria cells, Calcif Tissue Int 1983;35:578-585. 7 Canalis E, McCarthyT, Centrclla M. Isolation and characterizationof insulin-like growth factor I (somatomedin C) from cultures of fetal rat calvariae. Endocrinology 1988;122:22-27. 8 Frolik CA, Ellis LF, Williams DC. Isolation and chara~te~~tion of insulin-like growth factor II from human bone. Biochem Biophys Res C;>mmun 1988;151:1~11-1018.
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