Observations that IGF is produced and acts locally in multiple tissues raise important questions about the biological significance of the major pool of IGF present in the circulation. Does it represent a pool of endocrine IGF en route to the tissues or conversely growth factor produced in excess of autocrine/paracrine requirements undergoing elimination? The primary objective of this thesis was to examine the kinetics and distribution of circulating IGF in sheep with a view to determining tissue destinations and thereby potential functions of the blood borne hormone. The IGFBP play a central role in facilitating IGF action. Characterization studies of the IGFBP and an examination of their physiology and potential involvement in IGF transport are also important parts of this thesis. Such studies are necessary because potential therapeutic uses of IGF will depend on systemic administration and endocrine action. Early work involved structural/functional characterization of a batch of recombinant methionyl insulin-like growth factor-I (N-Met IGF-I) designated for this project. The peptide was heterogenous on reversed phase chromatography eluting as two major peaks of approximate abundance 1:2. These each had the amino acid constitution expected of N-Met IGF-I and were carefully characterized in a range of binding and biological assays. Whereas the early eluting peptide demonstrated much reduced activity in each assay system, the second peak proved equipotent to a highly purified ovine plasma IGF-I preparation and was chosen for the investigative work of this thesis. The early eluting peptide may represent a variant with mismatched disulphides. Initial characterization of IGF binding activity in ovine tissue fluids was performed by competitive IGF tracer binding techniques together with size exclusion chromatography (SEC) and IGF-I affinity chromatography. Binding proteins (BP) 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, was identified in adult sheep plasma, colostrum, follicular fluid and fetal sheep plasma, and may be the ovine equivalent of the soluble type 2 IGF receptor. A 150 kDa binding protein complex, of mixed specificity for IGF-I and II, was also identified. In addition to vascular fluids, the 150 kDa complex was identified in mammay 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 identified with distinct specificities for the IGF peptides. The BP 'make-up' of fluids and of 150 kDa and 40-50 kDa pools isolated by preliminary SEC was latter examined by IGF ligand blot analysis. Analysis of plasma 150 kDa pools revealed only the characteristic doublet of IGFBP-3 at 40-43 kDa, whereas the 40-50 kDa pool was heterogeneous containing IGFBP-3 together with smaller bands of 35, 30 and 23 kDa which may be the ovine equivalents of IGFBP-2, BP-4 and possibly BP-1. In support of the tracer binding data, IGFBP-3 was also identified in mammary lymph as were the smaller species. In an extension of the in vitro IGF tracer binding/SEC approach, kinetics of IGF equilibration with plasma binding sites was examined. Binding was found to be time and temperature dependent, reversible, dose responsive and relatively specific for the IGF peptides. Observations of special interest include a biphasic pattern of IGF-I equilibration with plasma, consistent with formation of the ternary 150 kDa complex of IGFBP-3, IGF and ALS, and evidence of relatively slow dissociation of IGF/BP complexes, suggesting that if release of IGF is required for full expression of IGF bioactivity in vivo, then specific processes may be involved. Avidity of isolated IGFBP complexes for Con A and heparin affinity adsorbents was also examined. The data indicate that the IGFBP belong to a relatively select group of proteins with high affinity for the glycosaminoglycan heparin suggesting roles for these proteins at the level of the capillary endothelium and/or extra-cellular matrix. Metabolic clearance of IGF-I and II was examined following intravenous (iv) bolus injection of the growth factors as radioiodinated tracer preparations. Tracer administration was followed by a rapid initial phase of clearance associated with tracer mixing in the vascular pool followed by intermediate and longer phases which appear to be direct consequences of interaction with and between the BP and to some extent accumulation of tracer degradation products in the circulation. Metabolic clearance of tracer complexed to the major molecular weight pools of BP was examined following SEC of sequential plasma samples. Average half-lives for IGF-I and II complexed to the 150 KDa and 40-50 kDa pools of carrier protein were established (150 kDa; 545±25 min, 325±30 min; 40-50 kDa, 34±2 min, 9.6±1.8 min, (mean±S.E.M., IGF-I and II respectively)) and compared to free IGF-I (t1/2 <5 min). Rapid clearance of free compared to bound IGF illustrates the central role of the IGFBP in maintaining IGF in the circulation and controlling tissue distribution. Whereas binding of IGF-II to different BP at 40-50 kDa (eg. IGFBP-2) may explain its shorter half-life compared with IGF-I, evidence suggests that IGF-I and II bind to the same carrier at 150 kDa. The observed difference in half-life of the 150 kDa complex is therefore suggestive of different metabolic handling of the BP depending on which of the IGFs is bound. The more rapid clearance of IGF-II complexed to the 150 kDa and 40-50 kDa carriers compared to IGf-I contributed to a more rapid clearance overall and is reflected in calculated metabolic clearance rates for IGF-I and II (IGF-I, 3.9 ml/min; IGF-II, 7.8 ml/min). Considering plasma IGF-II is significantly higher than IGF-I in post-natal sheep, a substantially greater secretion rate for IGF-II would be required to maintain plasma IGF-II in the face of the greater clearance rate. The secretion rate for IGF-II was estimated at ~ 1.6 nmol/min in the current study, some 8-fold greater than IGF-I. Clearance of IGF-I from plasma was associated with the appearance of radioactivity in lymph. Chromatography indicated that tracer in lymph was not degraded but retained its BP activity eluting on SEC complexed to high molecular weight BP. The data illustrate that blood borne IGF is distributed into the extra vascular space and may therefore be available to the tissues. This contention is supported by observations that relatively little radioactivity (<20% in the course of these experiments) was cleared from plasma into urine suggesting that plasma IGF is not principally an elimination form. Similarly no other significant sites of elimination were identified. Questions of how physiological control of the BP may influence tissue distribution of IGF were investigated in the next major experimental section of this thesis. In the first study the influence of nutritional manipulation and GH treatment of growing lambs on the molecular distribution of IGF immunoreactivity in plasma was examined using a new IGF-I RIA in conjunction with SEC and saturation analysis for the estimation of the BP. Total plasma IGF-I was found to increase with nutritional intake (P<0.01) and with GH treatment (0.25 mg/kg body weight/d; P<0.001) but only on the higher intakes. Molecular size fractionation revealed IGF-I immunoreactivity in 150 kDa and 40-50 kDa binding fractions. 150 kDa bound IGF-I was increased on the higher plane of nutrition(P<0.05) and by GH treatment (P<0.001) but again, only at higher levels of nutrition. By contrast no change in 40-50 kDa bound IGF-I was observed with treatment. Unbound IGF-I was also identified in sheep plasma (2-5% of total) but demonstrated only slight changes in relation to treatment. Saturation analysis was an analytical approach chosen to estimate total binding capacity (TBC) and relative saturation of the binding protein pools. Evidence suggests that in ovine plasma constituents of the 150 kDa complex are available in excess of endogenous IGF (P<0.001). Relative saturation of this species did not change with treatment despite the observed differences in 150 kDa bound IGF-I. The data suggest that components of the 150 kDa complex were themselves responsive to treatment. By contrast large differences in saturation of the 40-50 kDa species were observed (P<0.001) despite little treatment dependent change in bound IGF-I. Binding capacity of the 40-50 kDa fraction was elevated at low levels of nutrition and suppressed on the higher feed intake resulting in near saturation. The data indicate complex regulation of the IGFBP in sheep. IGF-I, elevated in response to higher nutritional intake and by CH treatment was mostly distributed into the 150 kDa complex; paradoxically the species which most effectively maintains IGF in the circulation. Thus in conditions presumably conducive with growth related processes (high GH, high nutrition) access of circulating IGF to the tissues is apparently most restricted. This evidence is difficult to reconcile with the view that 150kDa bound IGF represents a pool of endocrine IGF en route to tissue sites of action. Galactopoietic effects of GH in lactating ruminants appear to be exerted in the absence of a mammary GH receptor and are associated with increased plasma and mammary IGF-I content. Thus it has been proposed that blood borne IGF, acting in the classical endocrine fashion may be the mediator of GHs lactogenic effects. Consequently the lactating sheep surgically prepared by the catheterization of efferent mammary lymph may be a useful model for examining questions of IGF/BP physiology. In a further study, plasma and efferent mammary lymph concentrations of IGF-I were determined in lactating ewes before and after treatment with GH (10 mg/d) for 3 days. Analysis of paired plasma/lymph samples revealed that the capillary endothelium constitutes a barrier to the passage of macro molecules which reduces the concentration of IGF in lymph to ~ 35% plasma. A key observation from the current study was the GH dependence of mammary lymph IGF-I. Thus, GH was found to increase mammary lymph IGF-I concentrations by a proportionately greater amount than the increase in plasma IGF-I (P<0.01). The increase in lymph IGF-I resulted from an increase in the concentration of IGF associated with both high molecular weight (150 kDa) and low molecular weight (40-50 kDa) binding fractions. However, the data indicate a proportionately greater increase in 40-50 kDa bound IGF-I in lymph compared with plasma suggesting that treatment either induces a selective redistribution of plasma 40-50 kDa IGF and BP into the mammary gland or, alternatively, treatment increases intra-mammary production of these factors. Ligand blot analysis of mammary lymph revealed IGFBP-3 and -2 as the major constituents of this fluid. IGFBP-2 declined in lymph with GH treatment whereas IGFBP-3 appeared to increase. Additionally, saturation analysis indicated that a substantial proportion of lymph IGFBP-3 was present in the 'free', uncomplexed form. Consequently observations that the total binding capacity (TBC) of the lymph 40-50 kDa fraction increased with treatment, would appear to result from an increase in IGFBP-3. Total binding capacity of the lymph 40-50 kDa binding fraction was found to increase by a proportionately greater amount that its plasma equivalent. Thus, if the lactogenic effects of GH are mediated by IGF distributed from blood into the mammary gland, the mechanism by which it is transferred would appear to involve BP of the 40-50 kDa pool and in particular IGFBP-3. In the final experimental section of this thesis a novel system was employed to examine tissue distribution and destinations of blood borne IGF-I. This involved intravenous infusion of the N-Met analog of IGF-I together with specific immunologic detection. For this an IGF antibody was employed which recognises the recombinant N-Met variant but demonstrates minimal cross-reactivity with any of a range of other IGF peptides including ovine plasma IGF-I and recombinant authentic sequence IGF-I. This antibody can therefore recognise the N-Met variant against a background of authentic endogenous IGF-I and was usefully applied to examining tissue destinations of the N-Met variant following iv infusion. Plasma N-Met IGF-I rose to plateau concentrations of ~150 ng/ml during iv infusion (Infusion rate, 8 µg/kg/h). Analysis revealed the N-Met variant was distributed on plasma BP in much the same proportion as endogenous IGF. N-Met IGF-I immunoreactivity was identified in mammary lymph providing further evidence supporting the contention that blood borne IGF is distributed outside the vascular space. At the end of the infusion N-Met IGF-I was identified in all tissues examined contributing between 35% (in kidney) and 62% (spleen) to total IGF. Major differences in morphological distribution of blood derived (N-Met IGF) were revealed by autoradiography in post infusion tissue slices. Thus the N-Met IGF was found to contribute relatively little to total localizable IGF-I immunoreactivity in connective tissue elements of the samples examined (muscle and mammary) but, by contrast, blood derived (N-Met) IGF-I constituted ~85% of total IGF immunoreactivity in other tissues. In particular, these include the metabolically active regions of muscle and mammary (fibre and epithelium respectively). The evidence suggests therefore that fibre and epithelium may be targets for blood derived ‘endocrine' IGF. Differences in the abundance of blood derived IGF between tissues may relate to the accessibility (vascularization or capillary permeability) of specific tissues or, alternatively, to local rates of production and turnover of IGF in tissues. Thus the contribution of blood derived IGF to total localizable IGF may be expected to be less in tissues which actively synthesize IGF such as those in which autocrine/paracrine modes of IGF action are operative. Examples from the current study would be connective tissues of muscle and mammary. Conversely blood derived IGF would be expected to represent a greater proportion of total IGF in tissue targets for endocrine IGF. Support for the current data was obtained from IGF-I mRNA in situ hybridization studies performed on human fetal tissue (437). Stromal elements of muscle tissue (perimysium, epimysium) were identified as active sites of transcription as opposed to muscle fibre where message could not be detected. Thus the evidence suggests that the N-Met infusion model is a useful technique for delineating tissue targets for circulating (endocrine) IGF. It is now widely accepted that the primary actions of IGF on growth and development occur via autocrine/paracrine mechanisms close to its site of production. Nonetheless such arguments do not exclude the possibility of classical endocrine roles for the major pool of IGF present in the circulation. The primary thrust of this thesis has been to examine kinetics and tissue distribution of this pool of IGF. The data confirm the availability of blood borne IGF to extra vascular tissues and appear to indicate that it is distributed into the tissues on a selective basis and under physiological control. It may, therefore, be available to selected tissues to fill specific endocrine functions. / Whole document restricted, but available by request, use the feedback form to request access.
| Identifer | oai:union.ndltd.org:ADTP/275488 |
| Date | January 1991 |
| Creators | Hodgkinson, Steven Charles |
| Publisher | ResearchSpace@Auckland |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Whole document restricted but available by request. Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated., http://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm, Copyright: The author |
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