Passive immunization against insulin-like growth factor-I does not inhibit growth hormone-stimulated growth of dwarf rats.

Passive immunization against insulin-like growth factor-I (IGF-I) was undertaken in GH-deficient rats in an attempt to elucidate the relative importance of the endocrine vs.autocrine/paracrine actions of IGF-I in stimulating growth. Antiserum against IGF-I was raised in sheep and purified by affinity chromatography. The ability of the purified antibodies to neutralize the actions of IGF-I in vitro and bind IGF-I in vivo were extensively tested using L6 myoblast and cartilage bioassays. Four groups of male rats with isolated GH deficiency were used in the study. At 49 days of age the rats received 100 microliter normal saline given sc each day for 10 days, 2 mg/kg recombinant bovine GH (bGH) given in 100 microliter, sc, each day, 2 mg/kg bGH, sc, and 300 microliter immunoglobulin G purified from normal sheep serum given daily ip, or 2 mg/kg bGH plus 300 microliter anti-IGF-I immunoglobulin G daily, ip (a dose that was able to completely inhibit IGF-I actions on sulfate uptake into cartilage). Treatment with GH significantly increased growth rates (P less than 0.001) in the rats, but there was no difference between any of the three GH-treated groups; passive immunization against IGF-I did not diminish the GH-stimulated growth in these rats. Excess antibody could be detected in the plasma of all anti-IGF-I-treated rats at the conclusion of the experiment, and the antibody was capable of sequestering both free and binding protein-bound IGF-I. The absence of even a slight retardation of GH-stimulated growth in the anti-IGF-I-treated rats suggests that circulating IGF-I may not be important in mediating the growth-promoting actions of GH, although the immunoneutralization probably does not affect GH stimulation of tissue IGF-I production.

Distribution of circulating insulin-like growth factor-I (IGF-I) into tissues.

Intravenous infusions of amino terminal methionyl insulin-like growth factor-I (N-Met IGF-I; 8 micrograms/kg body wt x h; 24 h) were performed in lactating sheep and samples of mammary lymph, cerebrospinal fluid, and postinfusion tissues collected to examine distribution of the recombinant analog outside the vascular space. Samples were analyzed using an antibody specific for N-Met IGF-I and a second IGF-I antibody which recognized endogenous IGF-I and the N-Met variant equally. N-Met IGF-I infusion increased total plasma IGF-I immunoreactivity (ir) from 150 to 290 ng/ml. N-Met IGF-I was distributed into mammary lymph, increasing total lymph IGF-I from 60 to 130 ng/ml. By contrast iv N-Met IGF-I had no significant effect on IGF-I ir in cerebrospinal fluid. N-Met IGF-I was distributed on plasma and lymph IGF binding protein as endogenous IGF-I with binding to the 150,000 mol wt species predominant in plasma and the 40,000-50,000 mol wt pool of proteins predominant in lymph. N-Met IGF-I was also distributed into extra-vascular tissue accounting for 36% (kidney) to 62% (spleen) of total tissue IGF-I ir at the end of the infusion. The IGF-I antibodies were also used for the autoradiographical localization of IGF-I in postinfusion muscle and mammary tissue. No significant difference in antibody binding was observed to muscle fiber and mammary epithelium, but in marked contrast binding of the N-Met specific antibody to connective tissue of muscle and mammary was significantly less than the total IGF-I antibody (P less than 0.001; N-Met/total, 0.12). The data suggest that the contribution of blood-derived N-Met to total IGF-I varies markedly between tissues and provides evidence that blood-borne IGF-I may fill specific endocrine functions in selected tissues.

Presence of insulin-like growth factor-I receptors and absence of growth hormone receptors in the antler tip.

Red deer antler tips in the growing phase were removed 60 days after the recommencement of growth for autoradiographical studies and RRAs. Sections were incubated with radiolabeled GH or insulin-like growth factor-I (IGF-I), with or without excess competing unlabeled hormones, and were analyzed autoradiographically. There was negligible binding of [125I]GH in any histological zone of antler sections. [125I]IGF-I showed highest specific binding in the chondroblast zone to a receptor demonstrating binding characteristics of the type 1 IGF receptor. The lowest specific binding of [125I]IGF-I was to prechondroblasts. RRAs on antler microsomal membrane preparations RRAs on antler microsomal membrane preparations confirmed the absence of GH receptors and the presence of type 1 IGF receptors found by autoradiography. These findings suggest that IGF-I may act in an endocrine manner in antler growth through a receptor resembling the type 1 IGF receptor. The presence of type 1 receptors in the chondroblast zone implicates IGF-I involvement in cartilage formation through matrixogenesis. There is no support for IGF-I having a major role in mitosis in the antler.

Glycosaminoglycan binding characteristics of the insulin-like growth factor-binding proteins.

Interactions between the IGF-binding proteins (IGFBPs) and glycosaminoglycans (GAGs) such as heparin may be involved in the regulatory control of IGF exerted by the IGFBPs at the level of the extracellular matrix and capillary endothelium, although the precise mechanisms of this remain uncertain. We have searched primary sequences of human, rat and bovine IGFBPs-1 to -6 for putative GAG-binding consensus sequences (XBBXBX and XBBBXXBX, where B represents any basic amino acid and X is undefined). At least one such sequence was identified in each IGFBP examined except human and rat IGFBP-4 and rat IGFBP-6, with IGFBP-5 containing three GAG-binding consensus sequences. Additionally, the bovine IGF type II receptor was found to contain two such sequences in the intracellular region. Affinity of the IGFBP preparations for heparin was examined experimentally by affinity chromatography using pooled fractions of fetal and adult ovine plasma obtained by size exclusion chromatography. Pooled fractions of 150 kDa (containing IGFBP-3 alone by IGF ligand blot analysis) and 40-50 kDa (containing IGFBPs-3 and -2, together with proteins of 29, 24 and 25-28 kDa which may include IGFBP-4 and IGFBPs-1, -5 and -6) were found to bind strongly to the matrix necessitating high salt concentrations for their elution; however, in contrast, a > 200 kDa fraction containing the soluble form of the type II receptor failed to bind. Recombinant human non-glycosylated IGFBP-3 also bound strongly to the affinity adsorbent. No evidence of dissociation of bound IGF from binding protein complexes by association with the matrix was obtained from this experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

The ovine pancreatic protein which binds insulin-like growth factor binding protein-3 is procarboxypeptidase A.

Insulin-like growth factor binding protein-3 (IGFBP-3) is known to modulate the actions of insulin-like growth factors (IGF)-I and -II at the level of the cell. Proposed mechanisms include association of IGFBP-3 with cell surface proteoglycan, with cell surface binding proteins, proteolysis and/or internalization of IGFBP-3. In previous studies we have characterized a protein of 40 kDa in extracts of ovine pancreas and muscle which binds IGFBP-3 on ligand blot analyses. This paper reports the identity of the pancreatic species as procarboxypeptidase A (peptidyl-L-amino acid hydrolase, E.C. 3.4.17.1; proCPA). Identity was established by amino terminal sequence analysis, binding studies with pure bovine carboxypeptidase A (CPA) and observations that the binding activity was present in pancreatic secretions consistent with the role of proCPA as a secretory zymogen. The binding activity was inhibited by unlabelled IGFBP-3 at high doses (10 micrograms/ml) and reduced but not abolished by preincubation of 125I-IGFBP-3 with excess IGF-I. Digestion of 125I-IGFBP-3 with mature CPA produced a 26 kDa product. Modification of IGFBP-3 by CPA or binding to proCPA may provide a mechanism for modulation of IGFBP activity and hence IGF action.

Differential regulation of plasma levels of insulin-like growth factors-I and -II by nutrition, age and growth hormone treatment in sheep.

Plasma levels of IGFs-I and -II were measured in 4-month-old ewe lambs (n = 20) and 2-year-old ewes (n = 16), which were well fed (n = 18) or fasted (n = 18) for 3 days. Half of each nutrition group was given daily (0900 h) injections of bovine GH (bGH, 0.1 mg/kg body weight per day) for 3 days. Blood samples were collected immediately before the GH injection every morning. Plasma IGFs were extracted by acid gel permeation chromatography using a Waters Protein Pak 125 column, fitted to a Pharmacia fast protein liquid chromatography system, then freeze-dried, reconstituted (at pH 7.4) and estimated by RIA. At the end of the experiment, IGF-I levels in plasma were increased (P < 0.01) by exogenous bGH in both fed ewes and lambs but not in the fasted animals; plasma IGF-I levels were depressed by fasting (P < 0.01) at all ages. IGF-I levels were also found to be significantly higher (P < 0.01) in ewes than lambs. In contrast, plasma IGF-II concentrations were depressed (P = 0.02) by administration of bGH in all groups and elevated in the ewes (P < 0.05) by fasting. However, the lambs showed no significant changes in IGF-II with fasting. The IGF-II levels were significantly higher (P < 0.001) in lambs than ewes. Results from the present study demonstrate that GH administration stimulated an increase in plasma IGF-I and induced a decrease in plasma IGF-II. On the other hand, fasting depressed plasma IGF-I and elevated plasma IGF-II in the sheep. A significant GH/nutrition interaction for IGF-I (P < 0.01), but not for IGF-II, and a significant nutrition/age interaction for IGF-II (P < 0.01), but not for IGF-I, in the present study suggest that GH has a greater stimulating effect on plasma levels of IGF-I in the fed rather than fasted sheep and that nutrition has a greater influence on plasma levels of IGF-II in the older rather than younger animals, indicating that plasma IGFs-I and -II are differentially regulated by nutrition, GH and developmental stage in postnatal sheep.

Pharmacokinetics and bioactivity of intact versus truncated IGF-I during a 24-h infusion into lactating goats.

The aim of this study was to compare the plasma concentration profile, mammary blood flow response and transfer into milk of intact IGF-I with that of its truncated analogue, des(1-3)IGF-I (des-IGF-I). Each peptide was infused for 24 h into the pudic artery supplying one mammary gland of lactating goats (n = 5). Concentrations of IGF-I in plasma (from the jugular vein) rose rapidly during infusion of IGF-I or des-IGF-I to reach 510 +/- 62 and 640 +/- 32 ng/ml (mean +/- S.E.M.) respectively, compared with 262 +/- 35 ng/ml after a similar infusion of saline. Ligand blotting analysis indicated a significant increase in the intensity of [125I]IGF-I binding to the 40-43 kDa doublet (binding protein-3 (BP-3), P < 0.01) and the band at 31 kDa (P < 0.05) during infusion of either IGF-I or des-IGF-I, as compared with saline. Furthermore des-IGF-I induced a significant increase in intensity of binding to the 35 and 24 kDa bands, but IGF-I did not. Whereas [125I]IGF-I was distributed between BP-3 and the other binding proteins, [125I]des-IGF-I bound exclusively to BP-3. Mammary blood flow (MBF) increased 48 +/- 6% after 12 h of infusion of des-IGF-I, compared with an increase of 22 +/- 6% during IGF-I. The difference in response was significant at P < 0.05. In addition, more IGF-I was secreted into the milk of the infused than the non-infused gland during either infusion of IGF-I or des-IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Improved estimates of clearance of 131I-labelled insulin-like growth factor-I carrier protein complexes from blood plasma of sheep.

Clearance of protein-bound forms of insulin-like growth factor-I (IGF-I) from the circulation of sheep was determined using single injections of 131I-labelled ovine or [Thr59]-human IGF-I, in the 'free' form or prebound to 50 or 150 kDa plasma binding protein fractions. The half-life of circulating protein-bound forms of IGF-I was determined by size-exclusion chromatography of plasma samples taken over a 24- to 26-h experimental period. IGF-I bound to lower molecular weight binding protein(s) (approximately 50 kDa) showed a half-life of 26-40 min (mean 34 min; n = 6), while the half-life of a high molecular weight fraction (150 kDa) was considerably longer (range 398-603 min; mean 545 min; n = 8). Metabolic clearance of IGF-I following administration of free tracer ranged from 3.0 to 5.3 ml/min in sheep (n = 4) weighing 26.0-28.5 kg. Tracer distribution volume was 59 ml/kg liveweight (n = 4). Tracer degradation products were first detected in plasma 8 h after i.v. administration. No differences in stability of the purified ovine and recombinant human IGF-I tracer preparations were observed. However, a fraction of the [Thr59]-IGF-I tracer did not possess binding activity and this was associated with excretion of a greater proportion of administered radioactivity (over 22 h) in urine in animals receiving [Thr59]-IGF-I tracer (18.4-19.3%) compared with ovine IGF-I (7.1-11.0%).(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic clearance of insulin-like growth factor-II in sheep.

The metabolic clearance of ovine insulin-like growth factor-II (IGF-II) was examined in sheep using 131I-labelled IGF-II. Following i.v. administration the tracer was distributed in a volume similar to that of the vascular space (58.5 +/- 3.3 ml/kg; mean +/- S.E.M., n = 5) and demonstrated a triphasic pattern of clearance. Size-exclusion chromatography of a plasma sample collected 1 min after injection revealed peaks of radioactivity corresponding to hormone complexed to binding proteins of 150 and 40-50 kDa (relative abundance 21 and 65% respectively), a high molecular weight binding protein (greater than 200 kDa; 5%) and 'free' tracer (9%). Chromatography of sequential plasma samples revealed different patterns of clearance for these constituents. Half-lives of 131I-labelled IGF-II complexed to the 150 and 40-50 kDa binding proteins, as calculated from rate constants for their decay, were 351 +/- 30 and 9.6 +/- 1.8 min respectively (n = 5). These differ markedly from estimates for the clearance of IGF-I (545 +/- 25 min, n = 8, and 34 +/- 2.3 min, n = 6) associated with carrier proteins of the same apparent molecular weights. This was reflected in calculated metabolic clearance rates for IGF-I (3.9 +/- 0.5 ml/min) and IGF-II (7.8 +/- 1.0 ml/min). Chromatography also revealed that free IGF-II was reduced to negligible levels by 12 min. In contrast, radioactivity eluting in the position expected for the greater than 200 kDa binding protein was cleared from the circulation very slowly.(ABSTRACT TRUNCATED AT 250 WORDS)

Metabolic clearance rate of insulin-like growth factor-I in fed and starved sheep.

The metabolic clearance of insulin-like growth factor-I (IGF-I) has been examined in sheep using a radioiodinated hormone preparation (131I-labelled IGF-I). Following i.v. administration, 131I-labelled IGF-I was distributed in a volume equivalent to plasma (60 ml whole blood/kg liveweight) and demonstrated a triphasic pattern of clearance with apparent half-lives (t 1/2) of 4.0 +/- 0.4 (S.E.M.), 52.4 +/- 3.4 and 792 +/- 26.5 min (n = 10). No significant differences in the t1/2 of the three phases were identified in fed compared with starved animals (fed, n = 4, phase 1 = 3.1 +/- 0.64, phase 2 = 46 +/- 5.9 and phase 3 = 756 +/- 27 min; starved, n = 6, phase 1 = 4.6 +/- 0.58, phase 2 = 57 +/- 3.2 and phase 3 = 816 +/- 38.5 min). Similarly, no significant differences in the distribution volume (fed, n = 4, 44 +/- 4 ml/kg live-weight; starved, n = 6, 39 +/- 2 ml/kg liveweight) or metabolic clearance rate (fed, n = 4, 2.9 +/- 0.15 ml/min; starved, n = 6, 3.2 +/- 0.5 ml/min) of the IGF-I were found in fed compared with starved animals. High-performance gel filtration chromatography of sequential plasma samples following injection of 131I-labelled IGF-I revealed three clear peaks of radioactivity which demonstrated markedly different patterns of clearance. These correspond to hormone complexed to binding proteins of 150,000 and 50,000 daltons and to 'free' hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin-like growth factor-I protects myoblasts from apoptosis but requires other factors to stimulate proliferation.

Insulin-like growth factor-I (IGF-I) has been shown to stimulate myoblast proliferation for a limited time after which serum is required to reactivate IGF-I-stimulated myoblast proliferation. The aim of these studies was to determine whether IGF-I can stimulate myoblast proliferation and/or inhibit apoptosis alone or whether co-factors are necessary. This was achieved by investigating the proliferative response of L6 myoblasts to IGF-I and horse serum (HS) and by examining the status of cells in terms of cell number, substrate adherence, cell viability and DNA laddering following incubation with IGF-I and HS. L6 myoblasts proliferate in response to IGF-I after 36 h is not due to accumulation of waste products or lack of IGF-I. The addition of a low level (1% v/v) of HS restores the ability of myoblasts to proliferate in response to IGF-I and this supports the existence of a mitogenic competence factor. Furthermore, myoblasts failing to proliferate in response to IGF-I after 36 h regain the capacity to respond to IGF-I for a further period of 36 h when exposed to fetal bovine serum. Following the initial (36 h) phase of IGF-I-stimulated proliferation, removal of both IGF-I and HS led to a dramatic (60%) reduction in the number of cells fully attached to the culture vessel, with 60% of the completely detached cells dead. Agarose gel electrophoresis of extracts from these detached cells revealed higher levels of DNA laddering than extracts prepared from attached cells with IGF-I present. This suggests that IGF-I acts as a survival factor by protecting cells from apoptosis. In conclusion these experiments support the presence of a mitogenic competence factor in horse serum, which restores the ability of cells to proliferate in response to IGF-I. Unlike proliferation, protection against apoptosis is achieved by IGF-I or HS independently of each other.

Characterization of insulin-like growth factor binding proteins in ovine tissue fluids.

Competitive tracer binding studies using radioiodinated insulin-like growth factor-I and -II (125I-labelled IGF-I and 125I-labelled IGF-II) together with size exclusion chromatography and IGF-I affinity chromatography have been used to characterize IGF binding protein activity in ovine tissue fluids. Binding proteins of greater than 200, 150 and 40-50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus, the greater than 200 kDa binding protein, which is IGF-II specific, is present in plasma from mature sheep, colostrum and follicular fluid as well as fetal sheep plasma. This may be the ovine equivalent of the soluble type-2 IGF receptor recently identified in rat serum. The presence of a 150 kDa binding protein, of mixed specificity for IGF-I and IGF-II, in fetal and mature sheep plasma was confirmed in these studies. This protein, previously believed to be restricted to vascular fluids, was also identified in mammary lymph, follicular fluid and as a minor component in vitreous humor. Binding proteins of 40-50 kDa were revealed in every fluid tested and multiple variants with distinct specificities were also suggested. This was investigated by IGF-I affinity chromatography using mature sheep plasma. Following passage through the affinity adsorbent, binding of 125I-labelled IGF-I to proteins in the 40-50 kDa region was abolished but when 125I-labelled IGF-II was used as tracer, a binding protein of 40-50 kDa was still observed. Thus sheep plasma contains at least two 40-50 kDa binding proteins. The competitive tracer binding studies indicated that one such protein demonstrates mixed specificity for IGF-I and -II while the other strongly favours IGF-II.

Effect of growth hormone treatment on the distribution of insulin-like growth factor-I between plasma and lymph of lactating sheep.

Plasma and mammary efferent lymph concentrations of insulin-like growth factor I (IGF-I) were determined in lactating ewes before and after treatment with GH (10 mg/day) for 3 days. The lymph:plasma ratio of IGF-I increased from 0.34 to 0.47 after GH treatment when the IGF-I content of plasma increased by 19.4 nmol/l (from 32.1 nmol/l) and lymph by 13.7 nmol/l (from 10.7 nmol/l). This increase in the relative content of IGF-I in lymph was associated with increased lymph content of IGF-I in a lower molecular mass pool (nominally 50 kDa) derived by size exclusion chromatography. GH treatment increased the total binding capacity for IGF-I in both high (150 kDa) and low (50 kDa) molecular mass pools of plasma and the 150 kDa pool in lymph but there was a proportionally greater increase in 50 kDa total binding in lymph relative to plasma. Further, GH treatment increased the 'saturation' of the 50 kDa binding proteins but decreased the 'saturation' of the 150 kDa fraction, in both plasma and lymph. Ligand blot analysis of IGF-binding proteins (IGFBPs) in plasma and lymph showed that GH treatment of lactating sheep increased IGFBP-3 and decreased IGFBP-2 in plasma and lymph. Radioimmunoassay of IGFBP-2 showed that while GH treatment reduced the plasma content of IGFBP-2 by about half, the lymph:plasma ratio was increased from 0.68 to 0.87. GH treatment of lactating ewes not only increased the IGF-I content of plasma but increased the apparent efficiency of transfer of IGF-I across capillary endothelium to mammary efferent lymph.

Influence of nutrition and bovine growth hormone (GH) on hepatic GH binding, insulin-like growth factor-I and growth of lambs.

The effect on young lambs of 0.25 mg recombinant bovine GH (bGH)/kg per day on plasma concentrations of insulin-like growth factor-I (IGF-I), glucose, specific hepatic GH binding and body composition changes was examined at two levels of nutrition (lucerne pellets; 3 and 1.7% of body weight/day). Lambs on low levels of nutrition had low plasma IGF-I (P less than 0.001). Plasma concentrations of IGF-I were increased by bGH treatment at both levels of nutrition, with the high nutrition group showing the greatest IGF-I response after 3 and 40 days of bGH treatment. Plasma glucose, after 40 days, was higher overall (P less than 0.05) in lambs on high nutrition. bGH treatment increased plasma glucose, with the response being greater in the well-fed lambs. Specific binding of GH to liver membranes was highest in lambs on high nutrition and on bGH treatment; no significant interaction between nutrition and bGH treatment was detected, indicating that specific binding of GH was increased proportionally by bGH at both nutritional levels. The major change in body composition was the reduced level of fatness in lambs treated with bGH. There was no significant effect of bGH on body weight although bGH treatment tended to increase weight gain of well-fed lambs and decreased weight loss of poorly nourished lambs. The results show that, although there was a significant (P less than 0.05) bGH/nutrition interaction for IGF-I there was no such interaction for body weight/components or specific GH binding to the liver. The results indicate that an increase in plasma IGF-I does not necessarily result in increases in growth or changes in carcass composition.

Comparison of the effects of administration of recombinant bovine growth hormone or N-Met insulin-like growth factor-I to lactating goats.

Five goats were injected with GH (15 mg/day), three goats received systemic infusions of insulin-like growth factor (IGF)-I (43 nmol/h) and four goats received systemic infusions of physiological saline (20 ml/h) on days 4-6 of a 10-day experimental period during mid-lactation. Milk yield increased by an average of 24% in GH-treated goats by the time of the third injection. In contrast, milk yield of IGF-I-infused goats did not differ from saline-infused animals although two of three goats did show a small increase (12%) after 36 h of IGF-I infusion. With GH and IGF-I treatments plasma IGF-I concentrations increased similarly, reaching maxima of 100-130 nmol/l within 24 h. Plasma IGF-I concentration was relatively constant in saline-infused goats at about 50 nmol/l throughout the experiment. Total IGF-I bound to 50 kDa and 150 kDa binding proteins in plasma was increased by GH and IGF-I treatments but, in contrast to IGF-I, GH increased the proportion of IGF-I bound to 150 kDa binding protein. In a second experiment, four goats received systemic infusion of IGF-I (43 nmol/h) and four goats received systemic infusion of physiological saline (20 ml/h). There was no evidence that milk yield was changed during IGF-I infusion. However, when those goats which had previously received IGF-I infusions were injected with GH, milk yield increased by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of plasma and tissue levels of insulin-like growth factor-I by nutrition and treatment with growth hormone in sheep.

Tissue and plasma levels of insulin-like growth factor-I (IGF-I), and relative levels of liver IGF-I RNA, were measured in 6-month-old ewe lambs which were well fed (n = 10) or starved (n = 10) for 5 days. Half of each nutrition group was given daily (09.00 h) injections of human GH (hGH; 0.15 mg/kg body weight per day). Blood was sampled daily from 09.00 to 12.00 h at 15-min intervals through jugular vein catheters and the lambs were slaughtered 24 h after the fifth injection of hGH. Tissue and plasma IGF-I was extracted using an acid-ethanol-cryo-precipitation technique and estimated by radioimmunoassay. Tissue IGF-I was corrected for retained plasma IGF-I using tissue and blood hemoglobin levels. Liver IGF-I RNA levels were monitored by in-situ hybridization. Plasma IGF-I (nmol/l) was higher in both the fed group and the fed group given GH treatment. Tissue IGF-I from kidneys (nmol/kg) was also higher (P < 0.001) in the fed group. There was no significant difference in IGF-I concentrations in the muscle biceps femoris or liver between fed and starved lambs. Although GH treatment did not increase IGF-I levels in tissues significantly, IGF-I RNA levels in liver were increased (P = 0.02) in both fed and starved animals. The relative liver IGF-I RNA levels positively correlated with their corresponding tissue IGF-I levels in the fed group and the fed group given GH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Nutritional regulation of IGF-II, but not IGF-I, is age dependent in sheep.

In post-natal animals, plasma concentrations of IGF-I are tightly regulated by nutritional status. The current study reports that plasma levels of IGF-II in sheep are also regulated by nutrition, but whether plasma IGF-II is increased, decreased or remains the same, depends on the age of the animal. Ewe lambs, ranging in age from 2 days to 2 years, were fed or fasted for lengths of time between 24 and 72 h. Blood samples were taken at intervals of 24 h throughout the treatment period and immediately before slaughter. Plasma concentrations of IGF-I increased with advancing age in fed animals (P<0.001) and were reduced by fasting in all age groups (P<0.001). Plasma concentrations of IGF-II also increased as animals matured (P<0.001), but did not show an overall effect of the fasting treatment. An interaction between age and nutrition (P<0.001) resulted from a decrease in plasma IGF-II in response to fasting in neonatal animals (P<0.01) and, conversely, increased levels of plasma IGF-II in fasted mature animals (P<0.01 or P<0.001). Fasted sheep of peripubertal age showed no change in plasma levels of IGF-II. The nutritional sensitivity of serum IGF-binding proteins (BPs) also changed with age. The 29 kDa BP, which we presume to be BP1, was elevated by fasting in young animals and reduced slightly in older animals. BP2 was increased to a similar magnitude by fasting at all ages. BP3 was depressed by fasting in young animals and showed little change in adults. In contrast, a 24 kDa BP, which is probably BP4, showed little change in young animals and was reduced substantially in older sheep. In conclusion, the response of plasma IGF-II to fasting suggests that this peptide has functions in mediating nutritional stress which depend on the age of the animal, and also that the role of IGF-II may differ from that of IGF-I in adults.

Receptors for insulin-like growth factor-II in the growing tip of the deer antler.

Insulin-like growth factor-II (IGF-II) binding in the growing tip of the deer antler was examined using autoradiographical studies, radioreceptor assays and affinity cross-linking studies. Antler tips from red deer stags were removed 60 days after the commencement of growth, and cryogenically cut into sections. Sections were incubated with radiolabelled IGF-II, with or without an excess of competing unlabelled IGF-II and analysed autoradiographically. Radiolabelled IGF-II showed high specific binding in the reserve mesenchyme and perichondrium zones, which are tissues undergoing rapid differentiation and cell division in the antler. Binding to all other structural zones was low and significantly (P < 0.001) less than binding to the reserve mesenchyme/perichondrium zones. Radioreceptor assays on antler microsomal membrane preparations revealed that the IGF-II binding was to a relatively homogeneous receptor population (Kd = 1.3 x 10(-10) mol/l) with characteristics that were not entirely consistent with those normally attributed to the type 2 IGF receptor. Tracer binding was partly displaceable by IGF-I and insulin at concentrations above 10 nmol/l. However, affinity cross-linking studies revealed a single band migrating at 220 kDa under non-reducing conditions, indicative of the type 2 IGF receptor. These results indicate that, in antler tip tissues, IGF-II binds to sites which have different binding patterns and properties from receptors binding IGF-I. This may have functional significance as it appears that, whilst IGF-I has a role in matrix development of cartilage, IGF-II may have a role in the most rapidly differentiating and proliferating tissues of the antler.

Binding protein, radioreceptor and biological activities of recombinant methionyl insulin-like growth factor-I variants.

Reversed-phase chromatography (RPC) was used to resolve two variants of recombinant amino terminal methionyl residue (N-Met) insulin-like growth factor-I (IGF-I) with the same amino acid constitution but different disulphide linkages. Following radioiodination, equilibration with plasma and size exclusion chromatography at neutral pH the major form on RPC (approximate abundance 60%) demonstrated greater than 80% binding to 150 kDa and 40-50 kDa IGF binding proteins. This peptide has the RPC elution characteristics and disulphide assignment (Cys6-Cys48, Cys18-Cys61, Cys47-Cys52) of authentic with mismatched disulphides (Cys6-Cys47, Cys18-Cys61, Cys48-Cys52; N-Met IGF-I peak 1 peptide) demonstrated less than 15% binding under similar conditions. Potency of the peptides was investigated in competitive IGF-I plasma binding protein and L6 myoblast radioreceptor assays. The peak 2 peptide proved equipotent to purified ovine plasma IGF-I in each system but by contrast the peak 1 peptide was 40-fold and 200-fold less potent in the binding protein and radioreceptor assays respectively. Biological potency was examined in a non-competitive assay based on incorporation of [3H]leucine into confluent cultures of L6 myoblasts. In this system the N-Met IGF-I peak 1 peptide proved 15-fold less potent than the peak 2 peptide with correct disulphide linkages. Refolding variants may prove useful in establishing structure/function relationships for IGF-I.

Petrosal sinus catheterization in sheep. Development of a method for sampling hormonal output from the pituitary.

A relatively nontraumatic method has been developed to catheterize the petrosal sinus (PS) of sheep, via the internal jugular vein (IJV), using a percutaneous approach monitored by fluoroscopy. Preselection of suitable animals was facilitated by injecting radiopaque material through a cannula inserted into the deep facial vein to display the venous drainage from the pituitary. Further injections, via the same cannula, were later used to assist in the maneuvering of the catheter/wire guide combination as it passed up the IJV. To confirm catheter placement, plasma samples, collected simultaneously from PS and external jugular vein (EJV), were analyzed for growth hormone (GH). GH concentrations were consistently higher in the PS samples than in those found in the EJV, and more GH pulses were seen in PS samples than in the general circulation.