Abstract

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 μg/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.

Journal of Endocrinology (1996) 150, 51–56

Abstract

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.

Journal of Endocrinology (1995) 147, 507–516

Abstract

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. This difference between glands was greater (P<0·05) during des-IGF-I compared with IGF-I infusion, suggesting greater uptake of des-IGF-I by the gland compared with IGF-I, when infused locally. These findings indicate a greater bioactivity of des-IGF-I compared with IGF-I when infused locally into the mammary gland and may be explained by the different pattern of association of the two peptides with different binding proteins. The similar plasma profile and pharmacokinetics for IGF-I and desIGF-I during their 24-h continuous infusion of des-IGF-I or IGF-I is in contrast to results reported for a single injection of the peptides, probably relating to the ability of des-IGF-I to induce and bind to BP-3.

Journal of Endocrinology (1995) 144, 99–107

ABSTRACT

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 _½) of 4·0 ± 0·4 (s.e.m.), 52·4 ± 3·4 and 792 ± 26·5 min (n = 10). No significant differences in the t _½ 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 liveweight; 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. Highperformance 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. While radioactivity eluting in the position of free IGF-I had reduced to negligible levels by 12 min, ¹³¹I-labelled IGF-I associated with the intermediate molecular weight binding protein was still detectable 6 h after injection. In contrast, an initial increase in radioactivity associated with the 150 000 dalton binding protein was revealed. This form was cleared from the circulation very slowly, by 8 h it was the only species remaining and it was still detectable 26 h after injection. No significant differences in this pattern of bound and free hormone clearance were found in fed compared with starved animals. While there was apparently no interchange of hormone between the binding proteins in vitro the nature of the decay in vivo indicated the possibility of exchange of IGF-I between the different binding proteins.

J. Endocr. (1987) 115, 233–240

ABSTRACT

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 (>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 ± 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 > 200 kDa binding protein was cleared from the circulation very slowly. However, the small proportion of total radioactivity eluting in these molecular weight regions precluded calculation of decay constants for these species. Tracer degradation was monitored throughout the clearance study and estimated to be <20% at 800 min following i.v. administration. Less than 20% of tracer was cleared into urine over the 24 h of sampling, concurrent with a >90% fall in plasma radioactivity. Tracer in urine was completely degraded.

Journal of Endocrinology (1989) 123, 461–468

ABSTRACT

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%).

Following administration of free tracer or tracer bound to the 50 kDa protein, the proportion of radioactivity bound to the 150 kDa fraction increased over the first 20-30 min of observation. However, this was not apparent following administration of tracer bound to the 150 kDa protein, indicating that the more rapid turnover of IGF-I bound to the 50 kDa protein was associated, in part, with transfer of IGF-I to the 150 kDa binding protein fraction.

The calculated secretion rates of the IGFs were two- to threefold and twentyfold higher, for IGF-I and -II respectively, relative to that of insulin. These data are evidence supporting roles for IGFs in the regulation of metabolism.

Journal of Endocrinology (1989) 123, 469–475

ABSTRACT

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 haemaglobin 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. The lack of a significant IGF-I response to GH in tissues may be due to either the time at which tissues were sampled after the GH treatment or the dose of GH administered. However, the higher IGF-I concentrations in plasma and kidney from fed compared with starved animals and the positive correlations between liver IGF-I and IGF-I RNA levels suggest that tissue and plasma IGF-I is regulated by nutrition and GH, with nutrition playing a critical role in the regulation of tissue and plasma IGF-I in normal lambs.

Journal of Endocrinology (1993) 136, 217–224

ABSTRACT

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.

Journal of Endocrinology (1992) 132, 339–344

ABSTRACT

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%. While the increment in plasma IGF-I concentration during IGF-I infusion was similar to that obtained in the first experiment, the IGF-I response to GH injection was much greater, plasma concentrations rising to 230 nmol/l.

The data show a lack of milk yield response in goats whose plasma IGF-I concentrations were increased by IGF-I administration. Systemic infusion of IGF-I appears to be ineffective as a means of procuring a sustained increase in milk production.

Journal of Endocrinology (1989) 123, 33–39

ABSTRACT

Competitive tracer binding studies using radio-iodinated 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 >200, 150 and 40–50 kDa were revealed in these studies and shown to be widely distributed in body fluids. Thus, the > 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.

Journal of Endocrinology (1989) 120, 429–438