TY - JOUR
AU - Read, L., C.
AB - During early postnatal development, the intestine is highly responsive to LR3IGF-I administration but refractory to IGF-I, in contrast to the mature intestine. Given that LR3IGF-I is an IGF-I analog that binds poorly to IGF binding proteins, the response of the intestine is likely to reflect regulation of IGF-I bioactivity by IGF binding proteins. This study measures the delivery of exogenous IGF-I peptides to the intestine in preweaning (d-19) and adult rats to determine whether a correlation exists with the potency advantage of LR3IGF-I in the intestine during postnatal development. IGF-I or LR3IGF-I (2.6 μg/kg) was spiked with corresponding 125I-labeled peptide (10 × 106 cpm) and administered iv as a bolus (n = 5–6/group) with blood and tissue samples collected 5 and 10 min post injection. In both age groups, the levels of 125I-IGF-I retained in the blood at both 5 and 10 min were higher than the levels of 125I-LR3IGF-I, consistent with the slower clearance rate for the native peptide. In the gastrointestinal tract, the levels of 125I-LR3IGF-I per gram of tissue were 37–50% higher than 125I-IGF-I. Surprisingly, there was little difference in the relative delivery of LR3IGF-I to IGF-I to the intestine, across developmental age. Although bolus iv-injected LR3IGF-I was cleared more rapidly from the circulation than IGF-I and was subsequently delivered to the intestine in higher amounts than the native peptide, the ratio of LR3IGF-I to IGF-I in gut tissues was approximately 2:1 in both age groups. Hence, selective delivery to the gut is unlikely to explain the markedly higher potency of 125I-LR3IGF-I in stimulating growth of the preweaning vs. adult intestine. THE GASTROINTESTINAL TRACT is one of the most sensitive target organs for systemically infused IGF-I peptides (1–5). However, administration of the analog LR3IGF-I to adult rats results in a 3- to 5-fold increase in all growth and proliferative responses observed with the native peptide (4, 5). This potency advantage of LR3IGF-I is attributed to the differential regulation of the two peptides by the IGF binding proteins (IGFBPs). The affinity of the analog for the IGFBPs is markedly reduced because of the steric interference of the 13-amino-acid extension peptide on the N terminus coupled with a substitution of arginine for glutamate at the third position (6). Hence, the potent growth response of the intestine to LR3IGF-I, compared with IGF-I, implies that systemically derived IGF-I peptide action in intestinal tissues is regulated, at least in part, by the IGFBPs and that this regulation is in an overall inhibitory manner. Various lines of evidence indicate that this regulation is likely to involve prolonging the circulating half-life of IGF-I peptides (7–11) as well as tissue-specific actions regulated by IGFBPs produced at the local tissue level (12–14). In contrast to the adult gastrointestinal tract, the preweaning intestine responds very poorly to systemically delivered IGF-I (15). This refractory response does not seem to reflect a change in receptor numbers or changes to receptor signaling capacity because the preweaning intestine is highly responsive to LR3IGF-I despite the analog having a reduced affinity for the type 1 receptor, relative to IGF-I. Thus, LR3IGF-I elicits growth responses in the preweaning rat intestine, with over 20-fold greater potency than the native IGF-I, compared with a 3- to 5-fold difference in adult life. This experimental evidence suggests that the IGFBPs not only regulate systemically derived IGF action in the intestine but that the regulation differs across postnatal development. Our previous study examined the role of locally expressed IGFBPs in the proximal small intestine of the rat in regulating the actions of systemically administered IGF-I in the intestine (see Ref. 20). However, because the regulatory IGFBPs are also found in considerable amounts in systemic circulation, the current study examines the possible role of circulating IGFBPs in modulating the clearance and delivery of systemically administered IGF-I peptides to the intestine. The profile of circulating IGFBPs is well characterized and displays marked differences between suckling and adult rats: IGFBP-2 is the main species before weaning, at which time, IGFBP-3 rapidly increases, to become the predominant circulating species in adult life (16, 17). Moreover, the overall levels of IGFBPs in circulation markedly increase just before weaning, with the highest levels seen early in adult life. These differences in the profile and overall levels of circulating IGFBPs have important implications for the circulating half-life of IGF-I peptides and therefore the clearance of peptides from the circulation. For example, the circulating half-life of injected IGF-I increases from just 27 min in the circulation of neonatal rats to 3 h in the adult circulation (18). However, it remains unclear whether developmental differences in circulating profile of IGFBPs can explain the enhanced potency advantage of infused LR3IGF-I over IGF-I in the neonatal vs. adult gastrointestinal tract. We have examined this possibility by measuring the relative delivery of 125I-IGF-I, compared with 125I-LR3IGF-I, to the gastrointestinal tract in preweaning (d-19) and adult rats. To specifically examine the initial delivery of labeled peptides to the intestine from the circulation and not differences in retention of the peptides once delivered or changes caused by peptide degradation or secondary tissue distribution, samples were collected for measurement, at very early time points, after a bolus iv injection of either radiolabeled peptide into preweaning rats and adult rats. Materials and Methods Materials Recombinant human IGF-I and recombinant LR3IGF-I were purchased from GroPep Ltd. (Adelaide, Australia). Both peptides were iodinated with carrier free Na 125I (Amersham International, New South Wales, Australia) to a specific activity of 85 μCi/μg using the chloramine T method as previously described (19). Trichloroacetic acid (TCA) was from BDH chemicals (Victoria, Australia). Animals and animal maintenance Female hooded Wistar rats were obtained from the Adelaide University breeding colony. Preweaning rat pups were bred from the dams at the Women and Children’s Hospital. Experimental procedures in this study were approved by the Animal Care and Ethics committee of the Women’s and Children’s Hospital of South Australia. Animal handing and experimentation followed the Australian code of practice for the care and use of animals for scientific purposes. Rats were used at 19 d post partum (because the age group in which we have previously demonstrated the gut is refractory to IGF-I but highly responsive to LR3IGF-I) and at 90 d post partum (at which time, the gastrointestinal tract shows the adult pattern of responsiveness, with a LR3IGF-I:IGF-I potency ratio of only 3- to 5-fold). Experimental procedures The aim of the present study was to examine tissue distribution of radiolabeled peptides using a dose of iv-injected IGF-I or LR3IGF-I that is within the range (1–3 μg/kg·min) shown to stimulate growth of the rat gastrointestinal tract during long-term studies (3–5, 15, 20). Lyophilized IGF-I or LR3IGF-I was dissolved into 0.1 m acetic acid, then diluted to the appropriate concentration in PBS + 0.01% BSA, before the addition of 5–6 × 106 cpm of the corresponding 125I-labeled peptide. Rats at 19 d or 90 d post partum were assigned to one of the two peptide treatments. Each animal was gently restrained and lightly anesthetized by inhalation of Fluothane at a flow rate of 1.25 l/min (Halothane; ICI Pharmaceutical, Macclesfield, UK) before receiving the peptide infusate (2.6 μg/kg body weight) as a bolus via iv injection into the tail vein, using a 25-gauge butterfly-style cannula and Hamilton syringe. The dose of 2.6 μg/kg was delivered in a vol of 100 μl for preweaning pups (35–40 g) and 400 μl for adult rats (150 g), with the infusion timed to take 20–30 sec in each case. All animals were allowed to recover from anesthesia and were kept separate until the time of death, exactly 5 or 10 min later. Just before the time elapsing, the animals were reanesthetized to enable collection of blood via a cardiac bleed. Blood was collected for a maximum of 30 sec, immediately after the experimental time had elapsed. Animals were then killed by exsanguination, and a midline incision was made to expose and rapidly collect selected organs. Special care was taken to clean instruments between dissections to avoid radioactive cross-contamination. Organs removed for later analysis included the stomach, small intestine, colon, liver, kidney, and spleen. For both time points, there was a minimum of five animals in each peptide treatment group for each of the two ages examined. Whole blood and plasma (whole blood treated with heparin and centrifuged for plasma collection) were stored at −80 C for later analysis. Selected organs were weighed, snap-frozen in liquid nitrogen, and stored at −80 C to maintain radiolabeled peptide integrity until time of assay. The stomach and colon of preweaning rats were cleared of contents before weighing and freezing, but tissue fragility prevented this clearing of the small intestinal contents in tissue from this early age group. Hence, the intestinal contents were not removed from the tissue and were analyzed in conjunction with the tissue sample. The food contents were removed from all adult tissue samples before weighing and freezing. To reflect the tissue collection in the preweaning rats, the collected contents from the small intestine of adults were weighed, frozen, and analyzed for radiolabeled peptide, in conjunction with each corresponding small intestinal tissue sample. Experimental analysis Frozen tissue samples were homogenized on ice for several minutes at a ratio of 6 ml of ice-cold buffer (PBS + 0.1% BSA) per gram of tissue to normalize protein concentration of homogenates. Tissues homogenized included whole stomach, small intestine, colon, spleen, and kidneys (left only, for adult) with representative sub-samples of the liver (top right lobe) in both age groups; γ-radiation was measured in triplicate aliquots of tissue homogenates (500 μl), peptide infusate, and whole blood (25 μl each) to estimate total radioactive tracer in each sample. Aliquots were returned to ice before mixing with 25% (wt/vol) ice-cold TCA to give a final concentration of 10% (wt/vol) TCA. The peptide infusate, whole blood and tissue samples treated with TCA were kept on ice for at least 1 h and centrifuged, and the radioactivity in the TCA insoluble fraction was measured. An aliquot of each homogenized tissue sample was centrifuged (10,000 × g for 20 min at 4 C), and the absorbance of the supernatant was compared with that of whole blood at 405 nm to estimate the amount of residual blood from the relative heme content in each tissue segment (21). On subsequent analysis, the average radioactivity attributable to blood contamination of tissues as a percentage of total intact radioactivity in each tissue was less than 8% and 5% in preweaning and adult tissues, respectively. For each tissue sample, the radioactivity attributable to blood contamination was subtracted from the total radioactivity to determine the amount of intact tracer present in each tissue. The amount of TCA-precipitable radioactive IGF-I or LR3IGF-I per gram of tissue or per milliliter of whole blood was expressed as a percentage of the total infused TCA-precipitable radioactivity at the time of assay. All experimental analysis was conducted within a few days of the infusion experiments, to minimize 125I decay. The percent of peptide infusate that was TCA-precipitable at the time of experimental analysis remained high, at 85%, for IGF-I and 80% for LR3IGF-I. In blood, the percent of radioactivity that was TCA-precipitable ranged from 81–75% for IGF-I and 77–79% for LR3IGF-I at the 5- and 10-min time points, respectively. In tissue examined in this study, between 75 and 86% of 125I-IGF-I was TCA-precipitable in adult samples, with slightly lower levels in tissues from preweaning animals, ranging between 72–78%. In the case of LR3IGF-I tracer, the percent of TCA-precipitable radioactivity was 74–85% in adult tissues and 70–81% in tissues from the younger age group. Plasma samples were analyzed by neutral gel chromatography to determine the proportions of radiolabeled peptides in free form or bound to circulating IGFBPs. Plasma was thawed on ice, and 50-μl aliquots from animals within each treatment group were pooled. The pooled samples were mixed in an equal volume of freon (1,1,2-trichloro-1,2,2-trifluroethane; DuPont Australia, New South Wales, Australia) to remove lipids, and aliquots (120 μl) were subjected to size-exclusion chromatography using a Superose-12 column 1 × 30 cm (Pharmacia Upjohn, Uppsala, Sweden). The column was equilibrated and eluted in phosphate buffer [50 mmol NaH2PO4/liter, 150 mm NaCl/liter, 0.02% (wt/vol) sodium azide, pH 7.2]. The column was calibrated with molecular mass standards: human γ-globulin (150 kDa), BSA (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa) (Sigma, St. Louis, MO), and (100 μl) infusate solution of 125I-labeled IGF-I or LR3IGF-I (8 kDa). The flow rate was 1 ml/min, and 1.0-ml fractions were collected for measurement of γ-radioactivity, with absorbance of column elutate monitored at 280 nm to estimate protein elution. For each age group, circulating IGFBP profile was determined using pooled plasma from age-matched noninfused rats (2 μl/lane) subjected to SDS-PAGE and ligand blotting, as described previously (22), with exposure to x-ray film at −80 C for 6 d. Statistical analysis All data were reported as mean ± sem for a minimum of five rats per treatment group. Relative delivery of IGF-I to LR3IGF-I to various tissues was analyzed by one-way ANOVA using Student-Newman-Keuls post hoc test for all pairwise comparisons when significance was reached. P < 0.05 was considered significant. Results Distribution of injected IGF-I and LR3IGF-I peptides in the blood of preweaning and adult rats Five minutes after bolus iv infusion of radiolabeled IGF-I into preweaning rats, approximately 12% of the injected dose was recovered per milliliter of blood. The concentration in blood declined approximately 40% between the 5- and 10-min sampling times (Fig. 1). In comparison, the concentration of TCA-precipitable LR3IGF-I in blood was lower than IGF-I at each time point measured, with little change in blood LR3IGF-I concentration between the 5- and 10-min samples. The pattern was similar in adult animals, although the decline in IGF-I concentration between the 5- and 10-min sample times was lower (∼20%), suggesting a slower clearance rate from the adult circulation, compared with that in the preweaning circulation. Assuming the total blood volume is approximately 6% of body weight (23), we can estimate that, in both age groups examined, between 20–30% of IGF-I tracer was retained in the blood 5 min after bolus injection, compared with 10–15% of injected dose of LR3IGF-I, which is consistent with a faster clearance rate for the analog. Figure 1. Open in new tabDownload slide TCA-precipitable 125I IGF-I (black bars) and 125I LR3IGF-I (gray bars) recovered per milliliter of blood, expressed as a percent of the total dose injected into preweaning animals (left panel) and adult animals (right panel) at 5 or 10 min after bolus injection. Values represent mean + sem; n = 5–6 per group. Figure 1. Open in new tabDownload slide TCA-precipitable 125I IGF-I (black bars) and 125I LR3IGF-I (gray bars) recovered per milliliter of blood, expressed as a percent of the total dose injected into preweaning animals (left panel) and adult animals (right panel) at 5 or 10 min after bolus injection. Values represent mean + sem; n = 5–6 per group. Binding of exogenous IGF-I peptides with circulating IGFBPs in preweaning and adult rats Chromatography profiles of preweaning rat plasma showed approximately 80% of IGF-I tracer eluted at molecular mass values greater than 30 kDa, with most between the 29- and 150-kDa markers (Fig. 2). Both 5- and 10-min samples had similar elution profiles, with the exception that the 5-min sample contained a small peak that eluted earlier than the 150-kDa marker, possibly reflecting aggregates. We interpreted these results to indicate that, in preweaning rats, approximately 80% of the IGF-I recovered from the blood, at both 5 and 10 min after bolus injection, was bound to low-molecular-weight IGFBPs. The profile for the analog LR3IGF-I was different from that of IGF-I, with over 60% of the tracer eluting at the peak of free IGF-I (8 kDa), at either time point, in preweaning animals (Fig. 2). The bound LR3IGF-I tracer eluted at the position expected for binding to low-molecular-weight IGFBPs. Western ligand blots of preweaning rat plasma suggested that bound IGF-I and LR3IGF-I were most likely circulating with IGFBP-1 or -2, as indicated by the dominant band at 30 kDa, with very little binding at 45 kDa, the position at which IGFBP-3 elutes as a doublet on ligand blots (Fig. 3). Figure 2. Open in new tabDownload slide Neutral gel permeation chromatography of pooled plasma from 5–6 rats measured in samples collected 5 min (solid lines) and 10 min (dotted lines) after bolus iv injection of (A) 125I-IGF-I or (B) 125I-LR3IGF-I into preweaning (left) and adult rats (middle). The profiles show the recovery of radioactivity in 1.0-ml fractions as a percentage of total radioactivity eluted. The elution positions of γ-globulin (150 kDa), carbonic anhydrase (29 kDa), and 125I-IGF-I (∼8 kDa) are indicated. The far-right panels show the percent of each peptide eluted bound to plasma IGFBPs (>29 kDa) or eluting as free peptide (<29 kDa) in preweaning and adult rats, 5 min after injection. Figure 2. Open in new tabDownload slide Neutral gel permeation chromatography of pooled plasma from 5–6 rats measured in samples collected 5 min (solid lines) and 10 min (dotted lines) after bolus iv injection of (A) 125I-IGF-I or (B) 125I-LR3IGF-I into preweaning (left) and adult rats (middle). The profiles show the recovery of radioactivity in 1.0-ml fractions as a percentage of total radioactivity eluted. The elution positions of γ-globulin (150 kDa), carbonic anhydrase (29 kDa), and 125I-IGF-I (∼8 kDa) are indicated. The far-right panels show the percent of each peptide eluted bound to plasma IGFBPs (>29 kDa) or eluting as free peptide (<29 kDa) in preweaning and adult rats, 5 min after injection. Figure 3. Open in new tabDownload slide Circulating IGFBP profile in rat plasma, detected by Western ligand blot analysis, changes markedly during postnatal development. Lanes contained 2 μl pooled plasma from rats at d 19 (lanes 1–3) and d 90 (lanes 4–5), probed with 125I-IGF-II. 14C-labeled molecular-weight markers were run in the lane on the left (M). Figure 3. Open in new tabDownload slide Circulating IGFBP profile in rat plasma, detected by Western ligand blot analysis, changes markedly during postnatal development. Lanes contained 2 μl pooled plasma from rats at d 19 (lanes 1–3) and d 90 (lanes 4–5), probed with 125I-IGF-II. 14C-labeled molecular-weight markers were run in the lane on the left (M). In contrast to the profile of preweaning rat plasma, IGF-I in adult rat plasma eluted in a peak of approximately 150 kDa, with a smaller peak between the 29- and 150-kDa markers and only 15% eluting in a free form (Fig. 2). The 10-min profiles were similar to the elution profiles in the 5-min samples, except for a reduction in radioactivity across all peaks, consistent with the reduced recovery of total radioactivity in blood at this time point (Fig. 1). The LR3IGF-I profile in adult circulation differed substantially from that of IGF-I, with a higher percentage of tracer eluting as free peptide (∼30%); and, for the bound tracer, very little eluted at 150 kDa (Fig. 2). The results suggest that, in adult plasma, most of the IGF-I tracer is bound in the ternary complex with IGFBP-3 and the acid labile subunit, whereas LR3IGF-I, which has a lower overall affinity for the IGFBPs, is either free or bound to the lower-molecular-weight IGFBPs. The experimental design could not distinguish whether the LR3IGF-I binding was to IGFBP-1 or -2 or in binary complex with IGFBP-3. This interpretation is consistent with evidence from Western ligand blots, which showed that adult rat plasma contains substantially higher binding capacity, compared with preweaning rat plasma concomitant with the marked up-regulation of IGFBP-3 post weaning (Fig. 3). Delivery of radiolabeled peptides to the intestine of the preweaning and adult rats To compare the relative delivery of IGF-I and LR3IGF-I to the gut, the percentage of infused peptide recovered per gram of tissue was calculated for the stomach, small intestine, and colon. The data are expressed as a percent of the TCA-precipitable radiolabel in the infusate to normalize for any differences in the percentage of TCA-precipitability of LR3IGF-I, compared with IGF-I in the infusate at the time of assay. In the preweaning rats, 5 min post injection, 0.3–2.3% of injected IGF-I was recovered per gram of gut tissue, with an average of 1.6% per gram of tissue for the combined stomach, small intestine, and colon (Fig. 4, whole gut, left panel). Of all the intestinal segments examined, the small intestine showed the highest concentration of infused IGF-I. Ten minutes after bolus injection, the concentration of IGF-I tracer had increased to 0.75–3.8% of the total injected dose per gram of tissue, averaging 2.6% for the combined gut tissues (Fig. 4, right). The amount of LR3IGF-I per gram of tissue was approximately double that of IGF-I at either time point, averaging 3.5% and 4.4% for the combined gut tissues at the 5- and 10-min time points, respectively (Fig. 3). This indicates a markedly higher rate of delivery of LR3IGF-I than IGF-I to the gut of preweaning rats. Figure 4. Open in new tabDownload slide Levels of TCA-precipitable radiolabeled IGF-I (black bars) and LR3IGF-I (gray bars) per gram of gastrointestinal tissues of preweaning rats as a percentage of the total injected TCA-precipitable radioactivity at the time of assay. Tissue contents measured 5 min (left) and 10 min (right) after bolus iv injection. Values are mean ± sem for 5–6 animals in each group. All group comparisons of the relative delivery of IGF-I and LR3IGF-I were achieved by ANOVA. *, P < 0.05 vs. IGF-I in corresponding tissue. Figure 4. Open in new tabDownload slide Levels of TCA-precipitable radiolabeled IGF-I (black bars) and LR3IGF-I (gray bars) per gram of gastrointestinal tissues of preweaning rats as a percentage of the total injected TCA-precipitable radioactivity at the time of assay. Tissue contents measured 5 min (left) and 10 min (right) after bolus iv injection. Values are mean ± sem for 5–6 animals in each group. All group comparisons of the relative delivery of IGF-I and LR3IGF-I were achieved by ANOVA. *, P < 0.05 vs. IGF-I in corresponding tissue. In adult animals, IGF-I tracer was found throughout the gut, with an average concentration of 1.5% of the injected dose per gram of tissue at the 5-min time point (Fig. 5). As in the preweaning rat, the adult small intestine contained the highest concentration of all gut segments. However, in contrast to the findings in the younger age group, there was no change in the amount of IGF-I per gram in the adult gut between 5 and 10 min after injection. The amount of LR3IGF-I per gram of preweaning intestine was 16–45% higher than the amount of native peptide, depending on the region of the gut and the time of sampling. Overall, the amount of LR3IGF-I per gram of the combined gut tissue was 42% and 39% higher than that of IGF-I in the 5- and 10-min samples, respectively. Figure 5. Open in new tabDownload slide Levels of TCA-precipitable radiolabeled IGF-I (black bars) and LR3IGF-I (gray bars) per gram of gastrointestinal tissues of adult rats as a percentage of the total injected TCA-precipitable radioactivity at the time of assay. Tissue levels are measured 5 min (left) and 10 min (right) after bolus iv injection. Values are mean ± sem for 5–6 animals in each group. All group comparisons of the relative delivery of IGF-I and LR3IGF-I were achieved by ANOVA.*, P < 0.05 vs. IGF-I in corresponding tissue. Figure 5. Open in new tabDownload slide Levels of TCA-precipitable radiolabeled IGF-I (black bars) and LR3IGF-I (gray bars) per gram of gastrointestinal tissues of adult rats as a percentage of the total injected TCA-precipitable radioactivity at the time of assay. Tissue levels are measured 5 min (left) and 10 min (right) after bolus iv injection. Values are mean ± sem for 5–6 animals in each group. All group comparisons of the relative delivery of IGF-I and LR3IGF-I were achieved by ANOVA.*, P < 0.05 vs. IGF-I in corresponding tissue. To determine whether the preferential delivery of LR3IGF-I to the gut simply reflected enhanced clearance from circulation, we compared the relative amounts of radiolabeled IGF-I and LR3IGF-I delivered to other tissues known to be responsive to the growth-promoting effects of IGF-I (Table 1). In a striking difference from the pattern seen in the intestine, the ratio of LR3IGF-I to IGF-I in the nongut organs was approximately 1:1 in both preweaning and adult tissues, with the exception of a 2:1 ratio of LR3IGF-I to IGF-I in the adult liver and spleen measured 5 min after bolus injection of either peptide. However, this had reversed toward a 1:1 ratio by the 10-min time point, in agreement with the other tissues and the data for the preweaning tissues, indicating no selective delivery of LR3IGF-I to nongut organs measured in this study (Table 1). We conclude that the enhanced delivery of the analog to the gut reflects a tissue-specific response. Table 1. TCA-precipitable 125-I IGF-I and LR3IGF-I per gram of tissue of selected IGF-I-responsive target organs, 5 and 10 min after bolus injection 5 min 10 min IGF-I LR3IGF-I IGF-I LR3IGF-I Preweaning  Liver 7.1 ± 1.4 5.5 ± 1.7 7.0 ± 0.6 5.9 ± 0.7  Kidney 21.0 ± 4.4 26.3 ± 5.8 27.0 ± 3.7 24.6 ± 2.5  Spleen 2.9 ± 0.5 3.5 ± 0.7 3.2 ± 0.8 4.3 ± 0.5 Adult  Liver 0.88 ± 0.1 2.01 ± 0.5 0.88 ± 0.1 1.33 ± 0.2  Kidney 13.3 ± 1.9 16.6 ± 4.3 12.7 ± 1.3 14.9 ± 2.7  Spleen 0.51 ± 0.1 0.94 ± 0.02 0.5 ± 0.05 0.74 ± 0.1 5 min 10 min IGF-I LR3IGF-I IGF-I LR3IGF-I Preweaning  Liver 7.1 ± 1.4 5.5 ± 1.7 7.0 ± 0.6 5.9 ± 0.7  Kidney 21.0 ± 4.4 26.3 ± 5.8 27.0 ± 3.7 24.6 ± 2.5  Spleen 2.9 ± 0.5 3.5 ± 0.7 3.2 ± 0.8 4.3 ± 0.5 Adult  Liver 0.88 ± 0.1 2.01 ± 0.5 0.88 ± 0.1 1.33 ± 0.2  Kidney 13.3 ± 1.9 16.6 ± 4.3 12.7 ± 1.3 14.9 ± 2.7  Spleen 0.51 ± 0.1 0.94 ± 0.02 0.5 ± 0.05 0.74 ± 0.1 Data are expressed as a percentage of infused dose of radioactivity per gram of tissue. Open in new tab Table 1. TCA-precipitable 125-I IGF-I and LR3IGF-I per gram of tissue of selected IGF-I-responsive target organs, 5 and 10 min after bolus injection 5 min 10 min IGF-I LR3IGF-I IGF-I LR3IGF-I Preweaning  Liver 7.1 ± 1.4 5.5 ± 1.7 7.0 ± 0.6 5.9 ± 0.7  Kidney 21.0 ± 4.4 26.3 ± 5.8 27.0 ± 3.7 24.6 ± 2.5  Spleen 2.9 ± 0.5 3.5 ± 0.7 3.2 ± 0.8 4.3 ± 0.5 Adult  Liver 0.88 ± 0.1 2.01 ± 0.5 0.88 ± 0.1 1.33 ± 0.2  Kidney 13.3 ± 1.9 16.6 ± 4.3 12.7 ± 1.3 14.9 ± 2.7  Spleen 0.51 ± 0.1 0.94 ± 0.02 0.5 ± 0.05 0.74 ± 0.1 5 min 10 min IGF-I LR3IGF-I IGF-I LR3IGF-I Preweaning  Liver 7.1 ± 1.4 5.5 ± 1.7 7.0 ± 0.6 5.9 ± 0.7  Kidney 21.0 ± 4.4 26.3 ± 5.8 27.0 ± 3.7 24.6 ± 2.5  Spleen 2.9 ± 0.5 3.5 ± 0.7 3.2 ± 0.8 4.3 ± 0.5 Adult  Liver 0.88 ± 0.1 2.01 ± 0.5 0.88 ± 0.1 1.33 ± 0.2  Kidney 13.3 ± 1.9 16.6 ± 4.3 12.7 ± 1.3 14.9 ± 2.7  Spleen 0.51 ± 0.1 0.94 ± 0.02 0.5 ± 0.05 0.74 ± 0.1 Data are expressed as a percentage of infused dose of radioactivity per gram of tissue. Open in new tab Discussion We predicted that developmental differences in the profile and levels of circulating IGFBPs may influence the delivery of systemically administered IGF-I peptides to the gastrointestinal tract and thereby contribute to the massive potency advantage of LR3IGF-I, compared with IGF-I, in growth of the preweaning intestine. Hence, the relative delivery of 125I-IGF-I and 125I-LR3IGF-I to the intestine from the circulation of both preweaning and adult rats was measured. This study showed the analog was preferentially delivered to the gut of both preweaning and adult rats after a single bolus iv injection, compared with the native peptide. This differential was observed throughout the gastrointestinal tract from the stomach to the colon. Surprisingly, however, the relative delivery of the two peptides was similar in both age groups. Hence, selective delivery of the analog to the gut was unlikely to explain the marked potency advantage of LR3IGF-I in stimulating growth of the preweaning vs. adult intestine. In this study, LR3IGF-I was cleared from the circulation more rapidly than the native peptide after bolus iv injection into both preweaning and adult rats. This enhanced clearance of the analog was consistent with other experimental studies (15, 22, 24, 25) and attributable to the reduced binding affinity of the modified peptide with plasma IGFBPs (6), especially in the rodent (11). Indeed, a role for circulating IGFBPs in retarding the clearance of IGF-I peptides from the vascular space has been well established by a variety of experimental approaches, including the use of modified IGF-I analogs (as mentioned above) and combinations of IGF peptides and specific IGFBPs (10, 18, 26). Invariably, these studies show that IGF peptides or modified analogs that remain free in circulation are cleared more rapidly from the circulation, compared with IGFs that associate with IGFBPs. Our studies are consistent with these findings, in that the concentration of free LR3IGF-I in plasma was always greater than the percentage of free IGF-I. On the other hand, we were surprised that the current study revealed significant binding of LR3IGF-I to plasma IGFBPs, with 40–70% of the analog tracer eluting at higher molecular weights, by gel chromatography, in both preweaning and adult rats. The profile of IGFBPs that LR3IGF-I associated with differed from that of IGF-I, as shown by more IGF-I than LR3IGF-I present in the 150-kDa complex, by fast protein liquid chromatography analysis. Experimental evidence to support this interaction of the analog with IGFBPs in the rodent is limited; however, a recent study (24) also noted similar binding of LR3IGF-I to low-molecular-weight plasma IGFBPs in adult rats, although this other study did not address which particular IGFBP(s) were involved. Despite the differences in both the binding profiles and clearance rates of LR3IGF-I, compared with IGF-I, after bolus iv injection, seen in the current study, the increased clearance of the analog from the circulation, compared with IGF-I, was evident in both preweaning and adult age groups. Hence, it is unlikely that differences in the retention (or conversely, the rate of clearance) of the two peptides directly contributes to the age-related potency of these peptides in eliciting growth responses in the small intestine. LR3IGF-I was preferentially delivered to the intestine, compared with other tissues in the body, after bolus iv injection into both preweaning and adult rats. Although LR3IGF-I has an enhanced clearance from the circulation, specific tissue delivery of circulating peptides is not simply a function of the clearance rate from circulation. It remains unclear, with the available experimental evidence, how this preferential delivery of the analog is achieved. Because the analog bound to a different profile of IGFBPs in circulation, compared with IGF-I, we can postulate that the selective delivery of the analog to the intestine, compared with other tissues, may be influenced by IGFBPs in the various local tissue environments. More specifically, the endothelium and/or extracellular fluid of various tissues may contain very different total levels of IGFBPs present, different profiles of IGFBPs, and varying levels of specific receptors, all of which may have different degrees of saturation influenced by the presence of endogenous IGF-I or IGF-II (27–29). Each of these factors could potentially influence the potency of the native peptide, relative to that of the analog, and contribute to tissue specificity for IGF-I and LR3IGF-I actions. For example, the intestine is known to be rich in receptors, in comparison with other tissues (30), and may trap LR3IGF-I, which binds well to the IGF-I receptor, whereas IGF-I availability to these receptors may be reduced by interaction with local IGFBPs. To help explain the age-related response of the peptide in the intestine, these contributing factors would need to correlate to the responsiveness of the intestine with developmental age. Our previous study showed a decrease in the local expression of several IGFBPs present in the intestine with increasing developmental age (20), which could potentially influence the relative retention of IGF-I, compared with LR3IGF-I, in this tissue. This change may be further enhanced by IGFBPs present in the intestinal extracellular fluid, which are not reflected by local mRNA expression. In conclusion, iv injected LR3IGF-I was cleared more rapidly from the circulation than IGF-I, and the gut is the preferential site of delivery of the analog once it leaves the circulation. Notwithstanding the marked differences in the profile of circulating IGFBPs in preweaning, compared with adult circulation, the relative delivery of IGF-I to LR3IGF-I to the intestine was similar in both preweaning and adult rats. This is in contrast to the relative potency of the analog in stimulating gut growth, being markedly higher than IGF-I in preweaning rats (over 20-fold the potency), compared with that of adult rats (3–5 times as potent). Therefore, gut delivery of infused peptide in this short-term study does not correlate closely with biological potency seen in longer-term growth studies. 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TI - Preferential Intestinal Delivery of Long[Arg3] Insulin-Like Growth Factor (LR3IGF-I) over IGF-I in Preweaning and Adult Rats
JF - Endocrinology
DO - 10.1210/en.2002-220643
DA - 2003-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/preferential-intestinal-delivery-of-long-arg3-insulin-like-growth-egoCKR0yPu
SP - 1887
VL - 144
IS - 5
DP - DeepDyve
ER -