Insulin-like growth factor I (IGF-I) receptor overexpression abolishes the IGF requirement for differentiation and induces a ligand-dependent transformed phenotype in C2 inducible myoblasts.

Insulin-like growth factors (IGFs) stimulate both proliferation and differentiation of myogenic cell lines, and these actions are mostly mediated through the type I IGF receptor (type I IGF-R). To further investigate the role of this receptor in phenotypic characteristics of C2 murine myoblasts, we overexpressed the human type I IGF-R in the inducible clone of C2 cells, which requires IGFs in the differentiation medium to undergo terminal differentiation. Inducible myoblasts were transfected with either the eukaryotic expression vector pNTK or pNTK containing the human type I IGF-R complementary DNA, and we isolated two clones named Ind-Neo and Ind-R, respectively. Binding and autophosphorylation experiments indicate that Ind-R cells express about 10 times as much type I IGF-R compared with Ind-Neo control cells and that the transfected type I IGF-R is functional in Ind-R cells. We show that overexpression of the human type I IGF-R makes inducible myoblasts able to differentiate spontaneously, as assessed by expression of the myogenic transcription factors MyoD and myogenin, detection of the muscle-specific protein troponin T, and myotube formation. Moreover, when exposed to IGF-I, Ind-R cells lose contact inhibition, grow in the presence of a low level of growth factors and form colonies in soft agar, which is characteristic of a ligand-dependent transformed phenotype. It emerges from this study that 1) the type I IGF-R is strongly involved in the phenotypic differences between inducible and permissive cells with respect to the differentiation program; and 2) overexpression causes this receptor to act as a ligand-dependent transforming protein in muscle cells. We suggest that type I IGF-R abundance and level of activation may determine the efficiency of the autocrine mode of action of IGFs and discriminate their biological functions.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Secretion of insulin-like growth factors and their binding proteins by human normal and hyperplastic prostatic cells in primary culture.

Benign prostatic hyperplasia (BPH) is the most common benign proliferative disorder of unknown etiology found in men. Because insulin-like growth factors (IGFs) with their binding proteins (IGFBPs) are involved in the control of cellular proliferation, differentiation, and metabolism, we compared their secretion by prostatic epithelial and stromal cells in primary culture from the four different zones of normal prostate and from hyperplastic tissue to assess their contributions to the hyperplastic development. IGF-I could not be detected in the conditioned medium from either epithelial or stromal cells from normal and BPH tissues. IGF-II concentrations were the same in the conditioned medium from the epithelial cells of the different zones of the normal prostate and that of BPH cells. IGF-II concentrations secreted in stromal cell culture medium, however, were higher in the periurethral zone than in the peripheral and central zones. Moreover, in the periurethral zone, stromal cells secreted higher concentrations of IGF-II than did epithelial cells. Also, BPH stromal cells secreted more IGF-II than did BPH epithelial cells. IGFBP-3, IGFBP-2, and IGFBP-4 were all secreted by both epithelial and stromal cells. In contrast, IGFBP-5 was only produced by stromal cells of the periurethral zone of the normal prostate and BPH tissue. IGFBP-3 was predominantly secreted by normal stromal cells of the transitional zone. We observed that BPH stromal cells presented the same pattern of IGF-II and IGFBP production as normal stromal cells of the periurethral zone. These data support the hypothesis that the periurethral zone is the main region of the prostate implicated in the development of BPH. They also suggest that the variability in both IGF-II secretion and the secreted forms of IGFBPs, depending on anatomical location within the organ, may be important for the autocrine regulation of normal and hyperplastic prostate growth.

The insulin-like growth factor-II/mannose-6-phosphate receptor is present in fetal bovine tissues throughout gestation.

The insulin-like growth factor-II (IGF-II) and the IGF-II/mannose-6-phosphate (M6P) receptor are thought to play an important role in fetal growth and development. We have studied the expression of the IGF-II/M6P receptor in fetal bovine tissues from 5 through 36 weeks' gestation. Tissues from bovine fetuses were extracted in buffer containing 2% Triton-X-100 and 2% sodium dodecyl sulfate (SDS). Aliquots of the protein extracts were analyzed by SDS polyacrylamide gel electrophoresis and the protein bands were transferred onto nitrocellulose. Immunoblotting was performed with anti-bovine IGF-II/M6P receptor antiserum. In a subset of experiments, ligand blotting was carried out with radiolabeled IGF-II and subsequent autoradiography. IGF-II/M6P receptors were expressed in all tissues examined, with the highest amount of receptor being present in fetal lung and liver. Low amounts of receptors were measured in fetal brain. The amount of receptor was developmentally regulated throughout fetal life. The developmental regulation of receptor expression varied among the different tissues. In conclusion, the IGF-II/M6P receptor is present in all fetal bovine tissues examined. The presence of the IGF-II/M6P receptor seems to be developmentally regulated during bovine fetal life. We hypothesize that this receptor exerts important biologic effects during fetal growth and tissue and organ development.

Human IM-9 lymphoblasts as a model of the growth hormone-insulin-like growth factor axis: gene expression, and interactions of ligands with receptors and binding proteins.

Human IM-9 lymphoblasts bind growth hormone (hGH) and insulin-like growth factors (IGFs). We have systematically examined the IM-9 cells as a valuable model of the interaction of hGH and the IGFs at the cellular level. Cells were cultured in medium with 10% serum and for a subset of experiments cultured in serum-free medium. Binding of [125I]hGH and [125I]IGF-I and -II to intact IM-9 cells was measured: unlabeled hGH inhibited binding of [125I]hGH (half max. 20 ng/ml). Binding of [125I]IGF-I was inhibited by IGF-I (half max. 7.5 ng/ml), IGF-II (half max. 60 ng/ml), and insulin and anti IGF-I receptor antibody (alpha IR3). [125I]IGF-II was inhibited by IGF-II (half max. 15 ng/ml), IGF-I (half max. 500 ng/ml), insulin (half max. 250 ng/ml) but not by alpha IR3. Crosslinking experiments with [125I]IGF-II and DSS as the crosslinking agent and analysis of radioligand-receptor complexes by SDS-PAGE under reducing conditions revealed that [125I]IGF-II bound to a 250 kDa and a 135 kDa receptor species. The latter possibly represents an insulin-type receptor whereas the 250 kDa species had the characteristics of the IGF-II/M6P receptor. When IM-9 cell conditioned medium was analyzed in ligand blotting experiments with either [125I]IGF-I or -II a 30 kDa IGFBP species was detected on the autoradiographs. Also, IGF-II immunoreactivity (approx. 1 ng/ml medium) was measured in the cell conditioned medium using an IGF-BP blocked RIA employing [125I]IGF-II. In a subset of experiments IM-9 cells were homogenized in 4 M guanidinium-thiocyanate and RNA extracted in 5.7 M CsCl. Denatured RNA was electrophoresed on 0.8% agarose gels and transferred to a nylon membrane, fixed and the blots hybridized with cDNA probes. Probes were labeled with [32P]dCTP using a random prime labeling procedure: a Pst I 700 bp fragment of the human IGF-I cDNA, a 554 bp Pst I-Sal I fragment of the IGF-II cDNA, a 614 bp Pst I fragment of the IGF-I receptor cDNA and a 663 bp Pst I fragment of the IGF-II/M6P receptor. Autoradiographs of Northern blots showed specific hybridization with the IGF-I probe at 3.7 kb and with the IGF-II probe at 5.3 kb. No signal was detected with the IGF-I receptor cDNA probe. Hybridization with the IGF-II/M6P receptor probe yielded a 9 kb RNA species.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for a direct effect of growth hormone on osteoblasts.

In order to determine whether growth hormone (GH) exerts a direct effect on osteoblasts, in vitro and in vivo immunocytological studies were carried out on newborn rat calvaria and a clonal osteoblast-like cell line (MC3T3-E1) isolated from newborn mouse calvaria. After exposure to human growth hormone (hGH) or 1,25 dihydroxyvitamin D3 (1,25(OH)2D3), a significant increase in alkaline phosphatase activity was observed in MC3T3-E1 cells. Simultaneous exposure of MC3T3-E1 cells to hGH and 10 nM 1,25(OH)2D3 showed a synergistic effect of the two hormones on this activity. The optimal dose of hGH was 0.1 nM. An immunocytological procedure was performed on ultrathin frozen sections from 7-day-old rat calvaria and MC3T3-E1 cells cultured with hGH. GH-like immunoreactivity was observed in both cases. In calvaria, endogenous GH-like immunoreactivity was localized at the same ultrastructural level (plasma membrane, cytoplasmic and nuclear matrices) as exogenous GH-like immunoreactivity in MC3T3-E1 cells. Following the initial step of binding to the plasma membrane, GH may be internalized in the cytoplasmic matrix and nucleus. In situ hybridization revealed the presence of mRNA coding for GH receptor in calvaria cells. The density of these receptors seemed to be lower in osteoblasts than in hepatocytes. In MC3T3-E1 cells, hGH induced a dose-dependent secretion of insulin-like growth factor 1. In conclusion, these results indicate that GH may act directly on osteoblasts.

A developmentally regulated form of insulin-like growth factor receptor beta-subunit in C2 myoblasts exhibiting altered requirements for differentiation.

Ligand-dependent autophosphorylation and immunoprecipitation have been used to distinguish insulin and insulin-like growth factor-I (IGF-I) receptor beta-subunits in the permissive and inducible subclones of the C2 myoblast cell line. Permissive myoblasts differentiate spontaneously, whereas myoblasts of the inducible subclone require exogenous IGFs to undergo terminal differentiation. Permissive myoblasts contain beta-subunits of 95 and 101 kilodalton (kDa) mol wt. The 95-kDa subunits are immunoprecipitated with antipeptide antibodies directed against tyrosine kinase (AbP2), juxtamembrane (AbP4), and carboxy-terminal (AbP5) domains of the insulin receptor and insulin receptor monoclonal antibody 29B4. The tryptic phosphopeptide map of the 95-kDa band suggests that it contains both insulin and IGF-I receptor beta-subunits. The 101-kDa subunit is immunoprecipitated by AbP2, AbP4, and AbP5, because it forms a hybrid complex with the 95-kDa protein, but it does not react directly with AbP4, AbP5, or antibody 29B4. Phosphorylation of the 101-kDa subunit is more responsive to IGF-I than to IGF-II or insulin, indicating that it is a second IGF-I receptor beta-subunit. Inducible myoblasts exhibit a single major beta-subunit of 106 kDa mol wt. Its immunoreactivity and phosphopeptide map are virtually identical to those of the 101-kDa IGF-I receptor beta-subunit from permissive cells. However, unlike the 101-kDa beta-subunit, phosphorylation of the 106-kDa protein appears to be more responsive to IGF-II than to either IGF-I or insulin. It is lost upon differentiation of myoblasts into myotubes concomittant with the appearance of 95- and 101-kDa beta-subunits. These data demonstrate 1) an alpha 2 beta 2 IGF receptor that has high sensitivity for IGF-II in inducible, but not in permissive, myoblasts; 2) the beta-subunit of this receptor exhibits different migration in sodium dodecyl sulfate-polyacrylamide gels from either of those found in permissive cells; and 3) expression of this beta-subunit is developmentally regulated. This suggests that the inducible cell beta-subunit is a component of a stage-specific alpha 2 beta 2 IGF receptor subtype that functions as an IGF-II receptor.

Differentiation of adipocyte precursors in a serum-free medium is influenced by glucocorticoids and endogenously produced insulin-like growth factor-I.

Stromal vascular cells from rabbit perirenal adipose tissue differentiated at a high frequency in a chemically-defined serum-free medium containing insulin, transferrin, tri-iodothyronine and dexamethasone. The omission from the culture medium of dexamethasone resulted in a lack of adipose conversion. Addition of IGF-I increased glycerol-3-phosphate dehydrogenase (GPDH) activity. The conditioned media from adipocyte precursor cells contained measurable quantities of immunoreactive IGF-I as determined by RIA after neutralization of IGF binding proteins interference. Dexamethasone increased IGF-I secretion during the first seven days after plating and decreased IGF-I binding to conditioned media. Three molecular forms of IGF binding proteins (IGFBPs) were identified by Western ligand blots in conditioned media, with M(r) = 40,000, 29,000 and 25,000. The major form (M(r) = 29,000) was decreased by dexamethasone. In contrast, the M(r) = 24,000 form was increased. Specific binding of 125I-labelled IGF-I to rabbit adipocyte precursor cells was more effectively inhibited by unlabelled IGF-I than by unlabelled IGF-II or insulin. The electrophoretic migration of cross linked 125I-IGF-I to microsomal membranes revealed a complex with M(r) = 130,000 under reducing conditions corresponding to the alpha-subunit of the IGF-I receptor. The addition of IGF-I monoclonal antibody to rabbit adipocyte precursor cells cultured in serum-free medium significantly inhibited [3H]-thymidine incorporation and significantly decreased (50%) GPDH specific activities. This inhibitory effect was overcome by the addition of exogenous IGF-I. Thus stromal vascular cells isolated from perirenal adipose tissue secrete IGF-I and IGFBPs, possess IGF-I receptors and respond to exogenous and endogenous IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)

Functional and immunological distinction between insulin-like growth factor I receptor subtypes in KB cells.

Subtypes of insulin-growth factor I (IGF-I) receptors, including hybrid receptors containing insulin receptor alpha beta dimers associated with IGF-I receptor alpha beta dimers, have been described in a number of systems. The molecular basis of the multiple subtypes and their functional significance is not understood. Ligand-dependent phosphorylation of insulin and IGF-I receptors and immunoprecipitation with antipeptide and monoclonal antibodies have been used to characterize the subpopulations of these receptors in the human KB cell line. IGF-I receptors exhibit beta subunits of 95 and 102 kDa in these cells. IGF-I receptors containing 102-kDa beta subunits are immunoprecipitated by the IGF-I receptor-specific antibody alpha-IR3. Antibody alpha-IR3 does not appear to recognize a hybrid receptor in these cells. However, an antipeptide antibody against the carboxyl-terminal domain of the insulin receptor (AbP5) immunoprecipitates a population of receptors phosphorylated in response to IGF-I (1 nM) which contains both 95- and 102-kDa beta subunits. These receptors must be hybrid complexes because AbP5 does not recognize the 102-kDa beta subunit directly. The inability of antibody alpha-IR3 to recognize these complexes suggests that their IGF-I receptor alpha subunits must differ from typical IGF-I receptor alpha subunits either in primary sequence or conformation. Therefore, KB cells may contain more than one type of IGF-I receptor alpha subunit. Hybrid IGF-I receptors can also be distinguished from homotypic IGF-I receptors by their responsiveness to IGF-II. Stimulation of autophosphorylation in hybrid IGF-I receptors by IGF-I is 3-4-fold greater than that seen in response to IGF-II. In contrast, IGF-I and IGF-II are nearly equipotent in stimulating autophosphorylation in the alpha-IR3-reactive receptor population. This suggests the existence of functionally distinct receptor subtypes which may differ in their ability to mediate the biological effects of IGF-II.

GRF treatment of late pregnant ewes alters maternal and fetal somatotropic axis activity.

To examine the effects of anabolic agents given during late gestation on the maternal and fetal somatotropic axes, we injected pregnant ewes twice daily with 0.15 mg somatocrinin (GRF)-(1-29) for 10 days beginning on day 130 of gestation. Maternal and fetal endocrine changes were compared with control animals using both in vivo and in vitro approaches. Treatment with GRF increased maternal plasma levels of growth hormone (GH) and insulin-like growth factor I (IGF-I;P less than 0.05) but not IGF-II. Under in vitro test conditions, maternal pituitary cells showed a greater maximal response (P less than 0.001) to GRF. In the fetuses of treated ewes, cord plasma GH levels were not significantly increased compared with controls. These animals had similar IGF-I but higher IGF-II (P less than 0.05) plasma levels. The maximal response of fetal pituitary cells to GRF was increased (P less than 0.001). GRF treatment had no influence on maternal and fetal pituitary cell responses to somatostatin under either basal or GRF-stimulated conditions. In addition, these treatments did not affect plasma levels of placental lactogen, glucose, or free fatty acids in the maternal and fetal sheep. These data are compatible with the hypothesis that treatment of pregnant ewes in the last days of gestation with GRF could support accelerated fetal growth.

Application of preparative high-performance liquid chromatography to the purification of a fetal ovine insulin-like growth factor II: N-terminal sequence determinations using two different carriers.

A highly efficient procedure for the purification to homogeneity of an ovine fetal insulin-like growth factor II (IGF II) is described. Fetal sheep serum was used as the source material, and the bioactivity was followed throughout purification by an IGF II radioreceptor assay. Ovine IGF II was isolated by a combination of gel permeation, ion-exchange chromatography and reversed-phase high-performance liquid chromatography. The amino-terminal sequence of the first 36 amino acid residues was compared using two supports (polyethylenimine and polybrene) as carrier for protein sequencing. Ovine fetal IGF II was found to differ from human IGF II in three residues of the C-domain, with serine, isoleucine and asparagine substituted for alanine, valine and serine, respectively, at positions 32, 35 and 36. The final yield of highly purified ovine fetal IGF II was 134 micrograms, starting from 450 ml of serum.

Interactions of cathepsin-D and insulin-like growth factor-II (IGF-II) on the IGF-II/mannose-6-phosphate receptor in human breast cancer cells and possible consequences on mitogenic activity of IGF-II.

The lysosomal enzyme cathepsin-D (cath-D) and insulin-like growth factor-II (IGF-II), which share a common IGF-II/mannose-6-phosphate (M6P) transmembrane receptor, are both synthesized and secreted by breast cancer cells, upon which they might exert an intracrine/autocrine control on proliferation. We have evaluated the binding of 125I-immunopurified human cath-D in different breast cell membrane preparations. The concentration of high affinity M6P reversible binding sites (mean Kd, 0.85 nM) varied among the different breast cancer cells (0-0.82 pmol/mg membrane protein), but there was no correlation between the presence of steroid receptor and M6P-dependent binding. Cross-linking experiments with [125I]cath-D and [125I]IGF-II showed the formation of complexes with the 270,000 mol wt IGF-II/M6P receptor molecule which migrated, respectively, at 330,000 and 270,000 mol wt in 3-10% gradient sodium dodecyl sulfate-polyacrylamide gels. [125I]IGF-II cross-linking was increased by M6P (20% above control), whereas cath-D strongly inhibited IGF-II interaction by 80%. Conversely, IGF-II reduced [125I]cath-D cross-linking by 55%. Direct ligand binding on receptors transferred onto nitrocellulose sheets by Western blotting confirmed the interaction of both ligands on the same receptor molecule. By studying IGF-II's growth-promoting activity in these cells in a wide range of concentrations, we show that IGF-II triggers its mitogenic response via IGF-II/M6P receptor at low concentrations, whereas it is mainly acting via IGF-I receptor at high concentrations. Three lines of evidences lead us to that conclusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of a chronic GRF treatment on lambs having low or normal birth weight.

The effects of a long term treatment with human GRF(1-29)NH2 on plasma growth hormone (GH), somatomedin C (Sm-C), histomorphometric parameters of bone growth and body composition were investigated in normal and low birthweight male lambs. The animals were divided into two groups according to their birthweight: 24 normal birthweight (NBW) lambs weighing more than 4 kg and 22 low birthweight (LBW) lambs weighing less than 2.5 kg at birth. Half of the animals in each group received two daily subcutaneous injections (8 micrograms/kg body weight) of hGRF(1-29) NH2 (GRF) from birth to slaughter at 45 or 90 days of age. The other animals received the solvent only. At the beginning and at the end of the treatment, plasma GH and serum Sm-C concentrations were measured in all groups. After slaughter, a histomorphometric study was performed on undecalcified sections of metacarpal growth plates, and the remaining of the carcass was pulverized to study the chemical body composition. GRF induced GH release in both GRF-treated groups. However, plasma GH reached higher (P less than .001) concentrations and the GRF-induced GH peak lasted longer in LBW than in NBW lambs. At day 45, the GRF treatment increased (P less than .05) serum Sm-C concentrations in LBW. Most of histomorphometric parameters reflecting the metacarpal growth in length, were not statistically modified under GRF treatment. However, the size of degenerative cells was smaller (P less than .05) in LBW treated lambs as compared to controls. Consequently, the cell production in the growth plate was increased (P less than .05) under GRF treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Instantaneous secretion rate of growth hormone in lambs: relationships with sleep, food intake, and posture.

Relationships among sleep, feeding behavior, posture, and GH secretion were investigated in two groups of ruminant lambs in January (n = 6) and May (n = 3). Lambs were placed in individual cages and fed ad libitum. Behavioral features were obtained from continuous polygraphic recording. Blood was collected from undisturbed sheep every 3 min for 24 h via an indwelling catheter connected to a peristaltic pump. One month after the sampling session, ovine GH (oGH) was iv administered to evaluate oGH kinetic parameters. From GH plasma concentrations and oGH kinetic parameters, the instantaneous secretion rate of GH was reconstituted using a numerical deconvolution method. All lambs exhibited normal behavioral patterns. The clearance of oGH was similar for the two groups, and the daily production rates of GH were estimated at 14.60 +/- 7.99 micrograms/kg.24 h in January and 10.57 +/- 5.21 micrograms/kg.24 h in May. Analysis of concentration profiles indicate an episodic pattern of GH secretion into plasma. The mean number of peaks was 16.22 +/- 4.47/24 h, and the mean duration was 47.2 +/- 12.8 min for the nine sheep. When instantaneous secretion rates were taken into account, the number of identified peaks was similar, but the mean duration was reduced (32.9 +/- 9.8 min for the nine sheep). Significant relationships were not found between GH plasma concentration profiles and the state of vigilance, food behavior, or posture. Conversely, when the instantaneous secretion rates were taken into account, the highest GH production rate was detected during rest, i.e. slow wave sleep and rapid eye movement sleep, absence of food intake or rumination, and lying down. It is emphasized that the use of GH instantaneous secretion rate instead of GH concentration is of importance when evaluating the relationships between GH dynamics and short duration events. It is concluded that the influence of vigilance on GH secretion, which has already been demonstrated in humans, is likely to exist in other species.

Purification of chinook salmon (Oncorhynchus tshawytscha) GH for receptor study.

A method for the purification of chinook Salmon (Oncorhynchus tshawytscha) GH, which retains its biological activity, is described. The biological activity was investigated with an established radioreceptor assay using liver membranes from pregnant rabbits and bovine GH as standard and labelled hormone. The enrichment of the preparation was checked with electrophoresis (SDS-PAGE). Extraction and further steps were carried out using low molarity alkaline buffer (pH 8-10, M = 100 mM). Three chromatography steps were performed (Concanavalin-A sepharose, Bio-gel P60, DEAE). Ion exchange chromatography was performed under isocratic conditions (using a 50 cm column). Two isoforms (sGH1 and sGH2) were isolated. The purification yield is 0.7% compared to lyophilized pituitaries. The molecule is homogeneous in SDS-PAGE. Contamination by prolactin, gonadotrophin and corticotrophin is negligible (< 0.5%). It could be demonstrated that the biological activity of the preparation is maintained since this preparation stimulates the growth of juvenile trout (Salmo gairdneri) and binds specifically (35%) to trout liver membranes.

Photoperiodic control of growth hormone secretion and body weight in rams.

This study was undertaken to assess the influence of photoperiod on growth hormone (GH) secretion in rams and its possible influence on body weight. Twenty young adult rams were divided into two groups. One was subjected to an annual (AR) and the other to a semestral (SR) light regime during the same 18-month period. In both groups, daylength (DL) varied gradually between 8 to 17 hr. Plasma prolactin (PRL) and GH profiles consisting of 6 hr samples were determined and animals were weighed throughout the course of the experiment. Maximal PRL secretion was observed with largest DL. In contrast, GH secretion increased during increasing DL but it began to decrease before maximal DL was reached in both light regimes. Mean GH secretion was maximal when the DL was about 11 hr in SR and between 8 to 12 hr in AR. Similarly, body weight increased when DL increased and plateaued during decreasing DL in both AR and SR animal groups. Significant (P less than 0.05) differences were observed throughout the course of the experiment according to the effects of decreasing or increasing DL in each group. Analysis of variance showed that the effect of DL on plasma PRL and GH levels and weight velocity (WV) was significant (P less than 0.05) in both light regimes. This suggests that in SR, plasma PRL and GH levels and WV vary according to a six month period.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-binding proteins in hypophysectomized rat liver: characterization and subcellular localization.

We have characterized binding proteins for insulin-like growth factors (IGFs) in hepatic subcellular fractions and in the washed supernatants of these fractions in normal and hypophysectomized (hypox) rats. In the course of assessing IGF-II-binding sites on rat liver microsomes, we observed that [125I] IGF-II binding to the microsomal membranes of hypox rats was much lower than that in normal rats. Paradoxically, binding increased in hypox animals at low concentrations (0.5-5 ng/ml) of unlabeled IGF-II. After resuspension and centrifugation (washing) of the microsomes, no difference was found in [125I]IGF-II binding to hypox vs. normal microsomes. However, the binding of [125I]IGF-II to the washing supernatant (SN) from hypox rat microsomes was greater than binding to that from normal animals. Binding to SN was inhibited by unlabeled IGF-II in a dose-dependent manner. Scatchard analyses indicated that the affinity constant for binding by hypox SN was higher than that of normal SN and the microsomal fractions of both hypox and normal rats. After further subfractionation of the liver, no binding activity was found in SN from plasmalemma, whereas it was about 20% of input counts per min of [125I]IGF-II in SN from combined Golgi-endosome fractions of hypox rat liver. We next compared IGF-binding moieties in microsomal SN with those in plasma using cross-linking of [125I]IGF-II followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In normal rat plasma, we observed the presence of 42K, 39K, 31K, and 27K binding complexes. In hypox rat plasma only a 42-39K doublet was found. In the SN of normal rat microsomes, the predominant complex migrated at 39K and was distinguishable only after acidification. In the SN of hypox rat microsomes, the 42K complex was predominant, with a minor 34K complex. These studies have identified IGF-binding moieties in hepatic tissues, particularly in hepatic vesicular elements, which interfere in the binding of IGF-II to membrane receptors. Their presence in these receptor-rich elements may influence IGF binding to intracellular receptors and, hence, the biological activity of the peptide.

In vitro regulation of growth hormone (GH) release from ovine pituitary cells during fetal and neonatal development: effects of GH-releasing factor, somatostatin, and insulin-like growth factor I.

Using a monolayer approach, we have examined the acute (3 h) effects of GRF, somatostatin (SRIF), and insulin-like growth factor I (IGF-I) on GH release from pituitary cells of male and female 70-, 100-, and 130-day-old fetuses and newborn lambs and of prepubertal male lambs. GRF stimulated basal GH release in a dose-dependent (10(-12)-10(-8) M) manner at each stage in development. There was no linear relationship between maximal response and increasing age of the donor animals. The ED50 values for GRF were similar in all groups, except in the pituitaries from male and female 130-day-old fetuses, where the ED50 values were significantly higher. SRIF elicited a dose-related (10(-10)-10(-6) M) inhibition of basal GH secretion at each stage of fetal life and in the prepubertal period; although the response was lower in the youngest fetal pituitaries, there was no significant change in maximal response during the fetal or prepubertal period. No effect of SRIF on basal GH secretion was observed in newborn lambs. However, SRIF (10(-7) M) was able to block GRF (10(-8) M)-stimulated GH release in 100- and 130-day-old fetal and prepubertal as well as newborn lamb pituitary cells. Plasma IGF-I concentrations increased from 15.0 +/- 0.7 (mean +/- SE) and 13.8 +/- 0.9 ng/ml for male and female animals, respectively, at 70 days gestation to 55.8 +/- 3.2 and 51.8 +/- 11.1 ng/ml at the time of birth. The increase was much more pronounced in prepubertal lambs, especially in male animals, where IGF-I levels reached 300.8 +/- 37.7 ng/ml. IGF-I (100 ng/ml) had no effect on basal GH release in 70- and 100-day-old fetal, newborn, and prepubertal lamb pituitary cultures, but significantly inhibited basal GH secretion from 130-day-old fetal cells. This dose of IGF-I had no effect on GRF (10(-9) M)-stimulated GH release at 70 days gestation. It significantly inhibited this effect at 100 days and in prepubertal lamb cells. In 130-day-old fetal and newborn lamb pituitary cultures, IGF-I completely blocked the GH response to GRF.(ABSTRACT TRUNCATED AT 400 WORDS)