ABSTRACT

The measurement of insulin-like growth factors (IGFs) in plasma is complicated by the presence of high-affinity IGF-binding proteins (IGFBPs). Consequently, the IGFBPs need to be removed or their IGF-binding effects need to be neutralized prior to assaying samples for IGFs. It was observed that IGFs but not IGFBPs from sheep plasma bind to the size-exclusion gel Sephacryl S-100 HR at a low pH and that the IGFs can subsequently be eluted off at a neutral pH. From this observation a convenient method was developed for the extraction from plasma of ovine (o) IGF-I and -II free from detectable IGFBPs with close to 100% recovery. When assayed in homologous sheep IGF-I and -II radioimmunoassays the Sephacryl-extracted plasma samples gave dose–response curves which were parallel to purified oIGFs. Furthermore, the results obtained by Sephacryl extraction were highly correlated with those found by the established Sephadex G-75 extraction procedure.

Journal of Endocrinology (1993) 137, 239–245

Abstract

IGF-I was measured by RIA in plasma samples collected 8-hourly for 24 days which included two consecutive preovulatory surges of LH. In a separate study, ovarian venous blood was collected from animals undergoing ovariectomy on day 10 of the oestrous cycle, or 36 h later after being treated with prostaglandin with or without steroid-free bovine follicular fluid. Jugular venous blood samples were collected before, during and after surgery. Follicles were dissected from ovaries of these animals and sorted into categories of small, intermediate and large, non-atretic or atretic, and the follicular fluid was pooled and assayed for IGF-I. From another population of ovaries recovered from the slaughterhouse, granulosa, theca and corpora lutea were isolated, homogenized and assayed for IGF-I. Finally ovarian corpora lutea and granulosa cells were each incubated with tritiated amino acids overnight at 37 °C. Thereafter the tissues and media were sonicated, IGF-I extracted from the supernatant and tritiated IGF-I precipitated using a specific IGF-I antibody.

The absence of any significant change in peripheral IGF-I concentrations following ovariectomy and the finding that the ovarian venous IGF-I concentrations (161 ± 10 μg/l) were not significantly different from levels seen in peripheral blood (157 ± 10 μg/l) indicated that the ovary is not a net exporter of IGF-I. However, the ovary does synthesize IGF-I, as evidenced by granulosa and luteal synthesis, but probably not in quantities in excess of that utilized by ovarian tissues per se. Although the plasma IGF-I levels increased around the second preovulatory LH surge, the results overall indicated that the IGF-I concentrations in plasma are not strictly related to any major ovarian event during the oestrous cycle in the sheep. This view is based on the findings that the concentration of IGF-I in follicular fluid was not related to follicular health but correlated with those in peripheral plasma and that the ovarian venous concentrations did not vary between left and right ovaries irrespective of whether the ovaries contained a corpus luteum, dominant follicle or neither. Collectively, these results are consistent with the notion that IGF-I of ovarian origin fulfils an autocrine/paracrine function and does not have an endocrine role. Moreover, the results show that the concentrations of IGF-I in follicular fluid reflect those in peripheral plasma.

Journal of Endocrinology (1996) 148, 281–289

Abstract

Five lactating goats were infused, via an external pudic arterial catheter, directly into the mammary gland with 0·9% (w/v) NaCl (20 ml/h), recombinant human insulin-like growth factor-I (IGF-I; 80 nmol/h), recombinant human IGF-II (133 nmol/h) or IGF-I and IGF-II combined. The infusion was for 6 h and milk yield was determined every 2 h. The ratio of milk yield in the infused relative to the non-infused gland was changed only slightly by saline (2%), but increased to 9% (P<0·05) in response to IGF-I and 8% (P<0·05) in response to IGF-II. When combined, both peptides increased this ratio by 6%. These effects were elicited within 2–4 h of the beginning of infusion. Mammary blood flow increased 50–80% (P<0·05) during all IGF infusions, but only 28% during saline treatment. Plasma insulin decreased 50% (P<0·01) during the infusion of IGF-I alone or in combination with IGF-II and 25% in response to IGF-II alone. Whereas plasma glucose increased by approximately 10% during infusion of IGF-I alone or with IGF-II, it was not altered by infusion of IGF-II only.

The rapidity and unilateral nature of the milk-yield response to IGF-I and IGF-II is consistent with their acting directly on mammary tissue itself. Thus, the present results demonstrate similar local and systemic actions induced by intramammary infusion of IGF-II and IGF-I, although the magnitude of the response to IGF-II tends to be less than that to IGF-I.

Journal of Endocrinology (1994) 142, 93–99

Abstract

The putative negative feedback effects of IGF-I and IGF-II on GH secretion were tested by intracerebroventricular (icv) and intrapituitary administration to sheep. Over two consecutive days, serial jugular blood samples were taken at 10 min intervals for 6 h from ewes (n=3/group) fitted with indwelling stainless steel cannulae into the lateral or third cerebral ventricles. The sheep were injected (icv) with either vehicle or purified ovine IGF-I (2, 4 or 8 μg). IGF-I injection had no effect on plasma GH secretion. Serial blood samples were taken from a second group of nine ewes in which ovine or recombinant human (rh) IGF-I was infused (2·5 μg/h for 2 h) into the third ventricle; once again, IGF-I failed to affect the episodic pattern of GH secretion. Three ewes fitted with indwelling stainless steel cannulae placed in the anterior pituitary gland were consecutively infused with either ovine or rhIGF-I (2·5 μg/h for 2 h) or vehicle. Plasma GH concentrations were suppressed in 3/3 sheep from 1–1·5 h after the commencement of infusion and GH levels remained low for the remainder of the sampling period. In another group of five ewes synergistic effects of IGF-I and IGF-II on GH secretion were tested by icv infusion of rhIGF-I, rhIGF-II, or rhIGF-I+rhIGF-II (5 μg/h for 2 h) or vehicle (sterile 10 mm HCl/saline). Each sheep received each treatment in a randomised design. Infusion (icv) of IGF-I and IGF-II alone or in combination failed to alter GH secretion.

These observations suggest that IGF-I derived from peripheral tissues may modulate GH release at the pituitary level but that IGF-I acts neither alone nor in conjunction with IGF-II as a negative feedback regulator of GH secretion via the hypothalamus in the ewe.

Journal of Endocrinology (1995) 144, 323–331

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

The aim of this study was to examine the effect of the FecB^B fecundity gene on plasma concentrations and pituitary content of growth hormone (GH) in sheep. No differences were found between homozygous carriers (BB) and non carriers (++) of the FecB^B gene with regard to pituitary GH contents in both ovariectomized and intact ewes. However, ovariectomized ewes had higher levels of pituitary GH than intact ewes (P<0·01). There were no differences between FecB^B genotypes with respect to plasma concentrations of GH in 6-year-old ovariectomized ewes bled every 10 min for 12 h or in ram lambs bled weekly during their first year of life. GH levels in the rams decreased until week 27, increased to a peak at week 31 then decreased before increasing again at week 43. Mean plasma GH concentrations in the ewe lambs bled weekly for a year decreased until week 19 then remained at approximately this level for the remainder of the year. Mean GH plasma concentrations in the ram lambs were higher than in the ewe lambs (P<0·001). Ewe lambs that were homozygous for the FecB^B gene had lower body weights (P<0·05) and had higher levels of GH (P<0·01) than non carrier ewe lambs during their first year. Before the average age of first behavioural oestrus (36 weeks) GH levels in the ewe lambs were negatively correlated with body weights (r=−0·69, P<0·001, n=22). When body weight was included as a covariate in analysis of variance the genotype difference in ewe lamb plasma GH concentrations was no longer significant. In summary, pituitaries from ovariectomized ewes had higher levels of GH than those from intact ewes. There were no FecBB gene specific differences in pituitary levels of GH, the profile of plasma GH in 6-year-old ovariectomized ewes or in ram lambs during their first year of life. BB ewe lambs had higher levels of GH than ++ ewe lambs during their first year; however, this difference was probably due to the BB ewes having lower body weights than the ++ ewes because body weight was negatively correlated with mean GH levels.

Journal of Endocrinology (1995) 147, 217–223

Abstract

GH and IGF-I plasma concentrations were measured in lambs during an acute phase response induced by an intrathoracic injection of yeast. The acute phase response was indicated by reduced feed intake, weight loss and an increase in plasma concentrations of the acute phase protein haptoglobin. Intensive blood sampling on day 1 revealed elevated basal concentrations of GH in the yeast-injected group compared with concentrations in pair weight and ad libitum fed control lambs. This suggests that at the beginning of an acute phase response there is an increase in either GH secretion or the half life of GH. No evidence of a specific GH-binding protein in sheep plasma could be detected. IGF-I concentrations in the yeastinjected group remained constant for 3 days then increased to a peak level at day 6. In contrast, plasma IGF-I concentrations were depressed from days 3 to 6 in the pair weight control group and they were unchanged in the ad libitum fed controls. When the IGF-I concentrations were elevated in the yeast-injected group, this group had a higher daily weight gain despite their lower feed intake compared with the ad libitum fed controls. These results suggest that IGF-I may be associated with the increase in weight in the late stage of an acute phase response during recovery from an infection or injury. Day 1 GH peak amplitude concentrations in the yeast-injected lambs were negatively correlated with IGF-I concentrations on the following 2 days yet in the pair weight lambs the correlation was in a positive direction suggesting that the relationship between GH and IGF-I is different between animals that lose weight during an acute phase response and animals that lose weight because of feed restriction.

Journal of Endocrinology (1995) 144, 243–250