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.

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.

Oestrogen regulation of insulin-like growth factor binding protein-3 (IGFBP-3) and expression of IGFBP-3 messenger RNA in the ovine endometrium.

Transcriptional regulation of insulin-like growth factor binding protein-3 (IGFBP-3) by oestrogen was investigated by Northern blot hybridization of endometrial tissue mRNA from anoestrous ewes treated with oestradiol-17beta at either 0, 40 or 80 microg per day for 7 days. The 2.6 kb IGFBP-3 transcript, seen in control animals, was virtually undetectable in treated animals. This suppressive effect was reflected in Western ligand blots of the uterine luminal fluid (ULF) proteins where the concentration of IGFBP-3 was significantly decreased with increasing oestrogen treatments. IGFBP-2 levels were increased with oestradiol treatment but no significant effect was seen on the other minor IGFBP's present in the ULF. Northern analysis also showed that the IGFBP-3 transcript was present from days 12 to 16 of the oestrous cycle but was either absent or very weak on days 0 (oestrus) and 9 of cycling ewes. In situ hybridization of endometrial tissue sections localized the IGFBP-3 mRNA to the luminal epithelial cell layer, areas of the stromal tissue and in some glandular epithelial cells. Oestradiol treatment of ewes down-regulated expression of IGFBP-3 in the endometrium; therefore, the low levels of IGFBP-3 in ULF during the early part of the oestrous cycle is possibly due to elevated levels of plasma oestradiol around oestrus.

The proteolysis of insulin-like growth factor binding proteins in ovine uterine luminal fluid.

During days 12-15 after oestrus (day 0), the uterine luminal fluid (ULF) of both pregnant and non-pregnant ewes contains only two prominent insulin-like growth factor binding proteins (IGFBPs) of 16-18 kDa and 22-24 kDa which preferentially bind IGF-2. Immunoblotting with an IGFBP-3 antibody revealed these to be proteolytic fragments of IGFBP-3. In contrast, the ULF from anoestrus and ovariectomized ewes contained intact IGFBP-3 (40-44 kDa) and IGFBP-2 (34 kDa). Co-incubation of ULF from an anoestrus ewe with that from a day 12 cycling ewe cleaved the IGFBP-3 present into the two lower molecular weight IGFBPs characteristic of ewes in the late luteal phase of the oestrous cycle. The variation in proteolytic activity both during the year and during the cycle suggested an influence of progesterone. Supplementation of progesterone to long-term ovariectomized ewes via a CIDR-G breeding device for 5, 10 or 15 days induced marked proteolytic activity in all 10-day treated sheep. The ULF from the 15-day treated ewes showed reduced activity and could inhibit the activity present in 10-day ULF, suggesting the induction of an inhibitor after prolonged exposure to progesterone treatment. A possible role of IGFBP-3 proteolysis in the ovine ULF may be to selectively increase the bioavailability of IGF-1 in the uterine microenvironment, which may be crucial for the rapid elongation of trophoblast that begins during days 12-15 after mating.

Regulation by nutrition and age of insulin-like growth factor binding sites in ovine kidney.

The insulin-like growth factors (IGFs) are considered to have a role in the regulation of renal growth and development. The purpose of the present study was to evaluate the effect of nutritional stress on IGF binding in ovine kidney at different postnatal ages. Binding of IGF-I and IGF-II to kidneys of fed and fasted sheep was characterised using histological autoradiography, competitive binding assays, and SDS-PAGE. Nutritional regulation of IGF-I binding was restricted to cells of the proximal tubules of two and 14-day-old lambs where we identified an IGF binding protein which was upregulated in response to fasting and where IGF-II binding was also slightly enhanced. Ontogenetic changes occurred in the glomeruli where IGF-I binding peaked at 6 months (P < or = 0.001), and IGF-II binding increased to 4 months and then plateaued (P < or = 0.01). In the medulla, IGF-II binding was highest at 4 and 6 months (P < or = 0.05). From these studies, we conclude that the IGF axis may play a role in the regulation of the metabolic response to fasting in the kidney of young lambs. Furthermore, the changes with age which are described may reflect a transition period at 4-6 months, from an initial promotion of kidney growth and development in young lambs to establishment of the metabolic and clearance functions in the adult animal.

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)

Passive immunization of insulin-like growth factor (IGF)-1 and of IGF-1 and IGF-2 in chickens.

The effects of passive immunization against IGF-1 either alone, or together with immunization against IGF-2, on growth and metabolism were examined in chickens. Immunization against IGF-1 alone had no effect upon any aspect of growth, carcass composition, efficiency of energy utilization or hormone concentrations studied. Immunization against both IGF-1 and IGF-2 together resulted in a lighter final body weight (P < 0.05) compared with controls. Immunization against both IGFs together decreased abdominal fat content (P < 0.05) and resulted in a heavier mean spleen weight (P < 0.01). The joint immunization was also associated with elevated plasma T3 concentrations. These data may indicate a role for IGF-2, but not for IGF-1, in fat metabolism in chickens.

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)

Isolation and sequencing of deer and sheep insulin-like growth factors-I and -II.

We have developed a simple method for the isolation of highly purified cervine (c) and ovine (o) insulin-like growth factors-I (IGF-I) and -II. The IGFs were isolated from acidified serum by cation exchange chromatography and then purified by gel filtration, chromatofocusing, and reverse-phase chromatography. The IGF preparations are > 95% pure. The cIGF-I preparation contains < 0.056% cIGF-II and the oIGF-I preparation contains < 0.01% oIGF-II. Both the IGF-II preparations contain < 0.01% IGF-I. The amino acid sequence of cIGF-I has two differences when compared with human (h) IGF-I. The cIGF-II sequence, which is identical to bovine IGF-II, has three differences when compared with hIGF-II.

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.

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)

Evidence of a role for growth hormone, but not for insulin-like growth factors-I or -II in the growth of the neonatal rat.

Neonatal rats were injected with antiserum raised against either insulin-like growth factor (IGF)-I, IGF-II, rat growth hormone (rGH) or somatostatin (SRIF) on each of days 2-5 of life: controls received normal sheep immunoglobulin. Plasma levels of rGH and IGF-I were measured by radioimmunoassay and growth rates recorded. Neonatal administration of anti-rGH resulted in the suppression of plasma IGF-I levels at day 21 and of body weight gain compared with control animals from day 5 of age; relative growth velocity continued to diverge in the absence of any further treatment. Immunoneutralization of IGF-I or of IGF-II had no effect on growth rates of rats at any time during the experiment and had no effect upon plasma rGH concentrations at day 21. However, at day 7, plasma rGH was lower in anti-IGF-I-treated rats than in controls; in contrast, plasma rGH in anti-IGF-II-treated animals at day 7 was higher than in controls. Plasma levels of IGF-I at 49 days of age were similar regardless of the neonatal immunization treatment received. Anti-SRIF treatment of neonatal rats was associated with elevated rGH levels, but no significant stimulation of growth. These results indicated that growth hormone, but not circulating IGF-I or IGF-II are essential for normal growth in the neonatal rat.

On the Neuroendocrine Role of Insulin-like Growth Factors 1 and 2 in the Regulation of Growth Hormone Secretion.

It has been suggested that insulin-like growth factor-1 (IGF-1) may play a role in the regulation of growth hormone (GH) secretion. However, recent studies using recombinant IGF-1 contradict this. A possible role for IGF-2 has also been postulated, but data for this are fewer. In the present study we have sought to elucidate the role of IGFs in the control of GH secretion by central administration of both IGFs at near physiological doses. Further investigation of their physiological role in GH release has been studied using specific immunoneutralization of IGFs. Sheep with indwelling intracerebroventricular (icv) cannulae were given either IGF-1 (10 mug), IGF-2 (1 or 10 mug), or a combination of IGF-1 and IGF-2 (10 mug of each). Centrally administered IGF-1 had no effect on plasma GH, but IGF-2 caused a sustained decrease (P < 0.01) in plasma GH between 45 and 105 min after injection. The combination of IGF-1 and -2 resulted in a similar decrease in GH levels (P < 0.01). A potent immunoneutralizing antiserum against IGF-1 failed to have any effect on GH levels when administered icv, but administration of antiserum against IGF-2 stimulated GH release (P < 0.01) either alone or when administered together with anti-IGF-1 serum. These results indicate that central IGF-1 does not play a role in GH regulation. In contrast, central levels of IGF-2 may be physiologically important in regulating plasma GH levels.

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.

Neuroendocrine regulation of growth hormone secretion in sheep. V. Growth hormone releasing factor and thyrotrophin releasing hormone.

The effects of intravenous (IV) and intracerebroventricular (ICV) administration of either bovine growth hormone releasing hormone (GRF) or thyrotrophin releasing hormone (TRH) on plasma growth hormone (GH) and glucose levels have been examined in sheep. Intravenous GRF 1-29NH2 at 3 and 30 micrograms stimulated an increase in GH levels in a dose-dependent fashion; administration of GRF into a lateral cerebral ventricle, however, produced a smaller GH response which was similar at these two doses. Evaluation of somatostatin levels in petrosal sinus blood (which collects pituitary effluent blood) showed that ICV administration of GRF stimulated a release of somatostatin into the blood. Furthermore, concurrent administration of GRF and a potent anti-somatostatin serum ICV resulted in a much enhanced release of GH which was similar to that obtained with a comparable dose of GRF given IV. TRH (as another putative GH-secretagogue) was also administered both IV and ICV. When given IV, 200 micrograms (but not 100 micrograms) TRH produced an elevation in GH levels. By contrast, when 5 micrograms TRH was given ICV there was a decrease in circulating GH levels, but no change in plasma somatostatin concentrations. These results indicate that the smaller GH response to ICV- compared with IV-administered GRF is due to the release of somatostatin within the brain. In addition, it would seem that TRH is not a physiological GH-secretagogue in sheep.

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.

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.

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.