Insulin-Like Growth Factor-1 (IGF-1) is a potent mitogenic peptide synthesized by several cell types in mammals (1). IGF-1 has been implicated as a major factor stimulating growth and differentiation of many cell types during the perinatal period (2). IGF-1 has also been found in biologically relevant concentrations in milk from a number of species (3,4). In addition to endogenously produced IGF, a mounting body of evidence suggests that this milk-borne IGF plays a role in growth and development of the gastrointestinal tract of the suckling, and perhaps other tissues (5–6).
It is not clear whether biologically active IGF-1 is absorbed in significant quantities from the suckling intestinal tract (6–8). Some studies have demonstrated either absorption of receptor-active IGF-1 or increased peripheral blood IGF levels after enteral IGF administration in the suckling (9–10). In a recent report (7), receptor active 125I-IGF-1 was found in significant amounts in portal blood of infant rats after a single enteral feeding. The amount recovered was estimated as 20 to 30% of the dose administered. Interestingly, mother-fed calves have higher serum IGF-I levels than do animals fed an artificial milk devoid of growth factors (9). These results have now also been confirmed in piglets (11) and in adult rats (12). In a recent study, serum IGF-1 levels in adult men correlated with the intake of dairy foods, particularly milk (13).
The mechanism by which IGF-1 might be absorbed by the gastrointestinal tract is currently unknown. In vitro studies have suggested that if absorption does occur, it is not necessarily receptor mediated (14,15). The present in vivo study was performed in suckling rats to test the hypothesis that enteral absorption of this growth factor is nonsaturable.
rhIGF-I was obtained from Pharmacia and Upjohn Biosciences Center, Stockholm, Sweden, (Mats Lake, Ph.D.). 125I was purchased from New England Nuclear (Boston, MA). Bovine serum albumin, trichloroacetic acid (TCA), phenylmethyl sulfonylfluoride (PMSF), Sephadex G-50, and chloramine-T were obtained from Sigma (St. Louis, MO).
Animals, Surgery, and Experimental Design
Sprague-Dawley rats were obtained from the rat colony at the University of Arizona Health Sciences Center. Litters were culled to 10 pups by 2 days of age and kept with their dams until time of study. On the morning of each experiment, rat pups (age 10–12 days) were separated from their mothers and fasted for between 4 and 6 hours before study. Animals were kept in a breeding cage which was partially situated over an electric heating pad to maintain body temperature. All rats were weighed and anesthetized using subcutaneous ketamine HCl (Ketaset, Aveco, Fort Dodge, IA), xylazine (Anased, Lloyd Laboratories, Shenandoah, IA), and acepromazine maleate (PromAce, Aveco) in doses of 25, 5, and 1 mg/kg body weight, respectively. Animals were placed on warming pads to maintain body temperature during the experiment. A midline abdominal incision was then made and a 4-cm length of jejunum localized in situ. The intestinal segment to be studied was further isolated using 10-mm microclips (Roboz Surgical Instrument Co., Rockville, MD), taking care not to disrupt bowel integrity or blood flow.
Rat pups were paired temporally so that control animals received an intra-intestinal loop injection (27 gauge butterfly-style cannula (Abbott Laboratories, Chicago, IL) of 125I-IGF-1 (approximately 1–2 x 105 cpm or 0.25–0.5 ng IGF-I) in 0.1 ml of TRIS-HCl buffer (pH 7.4). Paired littermates received similar doses of 125I-IGF-1, but were pretreated 20 minutes before with nonradioactive rhIGF-I. These paired animals (“preincubated group”) were randomly given intraluminal loop injections of nonradioactive rhIGF-1 in concentrations of either 20, 500, or 1000 ng/ml (0.1 ml in TRIS-HCl buffer). These doses were chosen to represent, respectively, the concentrations of IGF-I in mature rat milk, colostrum of several species, or a representative pharmacological dose (3,4,9). Twenty minutes after the 125I-IGF-1 intraluminal injection, all animals were killed by decapitation and the livers and intestinal loops removed for further study. The experimental protocol was reviewed and approved by the Animal Care Committee of the University of Arizona.
rhIGF-1 was iodinated using the chloramine T method as previously described (16). After iodination, the peptide was further purified using gel chromatography (G-50 Sephadex) via a glass column (1.2 × 55 cm). Fractions were subsequently tested for specific binding to a crude bovine placental membrane preparation bearing Type 1 IGF receptors (17). Fractions exhibiting the highest specific activity were pooled and used within 3 and 5 days of iodination.
Sample Preparation and Assessment
Livers were placed in individual vials after blotting and weighing and subsequently assessed for radioactivity using a Packard AutoGamma gamma counter (Packard Instruments, Palo Alto, CA). Intestinal loops were dissected from the remaining intestine and flushed with 0.5 ml TRIS buffer and 0.5 ml air. Luminal fluid and loop specimens were counted separately. For each specimen, the radioactivity observed was then expressed as a percent of the labeled IGF-1 cpm injected using the following formula:
To compare the results of different pretreatment concentrations of IGF-1, the differences in retained radioactivity from jejunum or liver between controls and littermates pretreated with unlabeled IGF-I (preincubation groups) were calculated by assigning the control tissue cpm a value of 100% using the following formula:
Differences were then also compared between the three IGF-1 pre-incubation dosage groups.
To determine whether radioactivity observed in intestinal loop specimens was “intact,” peptide, intestinal loops, and livers were then homogenized in equal weight/volume of TRIS-HCl buffer with 0.2 M PMSF using an electric homogenizer. Homogenates were subsequently added (equal volumes) to a solution of 20% TCA and incubated for 30 minutes. Radioactivity of the mixture was measured, the samples centrifuged at 3000 rpm for 15 minutes, 40 C, the precipitate decanted, and the remaining radioactivity assessed. The percent radioactivity remaining in the precipitates represents an approximation of the ratio of radioactive peptide to total specimen radioactivity, which was confirmed in a previous study (16). To verify this latter point, however, selected intestinal homogenates were also subjected to gel chromatography (G-50 Sephadex, glass column 12. × 55 cm) after addition of acetic acid to bring the mixture to 0.1M. The positions of the eluted radioactive peaks were compared to that of the purified injected 125I-IGF-1 as previously described (16). Liver homogenates exhibited lower levels of radioactivity and were not chromatographed.
Data are represented as mean ± SE M. Differences between controls and littermates pretreated with different doses of IGF-I were assessed utilizing analyses of variance (ANOVA) and post hoc paired Student's t tests. Linear regression analysis used the least squares method. ANOVA, t tests, and linear regression analyses were performed with a computer (MacIntosh 8600/200 Power PC, Apple, Cupertino, CA) and statistical analysis software (Statworks, Cricket Software, Malvern, PA and Statview, Abacus, Berkeley, CA).
A total of 4 litters were used for the paired intestinal loop incubation experiments and an additional 2 litters were used for the TCA precipitation and chromatographic studies. After incubation with 125I-IGF-1 alone, intestinal loops (exclusive of luminal fluid) retained between 10 and 15% of the radioactive dose administered after the 20-minute incubation period. (See formula (1) in Methods for % of dose-administered calculations). As depicted in Table 1, pre-incubation with unlabeled IGF-1 was associated with a biphasic retention pattern of 125I-IGF-1 whereby increasing pre-incubation concentrations of unlabeled IGF-1 were associated with increasing retention of radioactivity. Thus, animals whose jejunal loops were pre-incubated with 1000 ng/ml IGF-I retained more than 3% more of the administered IGF radioactivity than their respective controls (P < 0.05 from results of 20 or 500 ng/ml experiments).
Radioactivity retained in liver specimens from rat pups was much lower than intestinal segments studied and was between 1.5 and 2.0% of the dose administered. When these results were compared to paired specimens after pre-incubation with unlabeled IGF-1, a biphasic pattern of retention of radioactivity similar to that observed in the intestinal segments was noted. No statistically significant differences between paired control and pre-incubation animals were observed, however, nor between animals with different pre-incubation IGF concentrations.
These results are more clearly represented after standardization by arbitrarily assigning all the control animal loops as 100% of retained radioactivity. (See formula (2) in Methods). In Figure 1A (jejunum), it may be noted that a progressive increase in cpm retained is present, with the value at 1000 ng/ml significantly higher than at 20 ng/ml (P < 0.01). A similar pattern was noted for liver (Fig. 1B), but no statistically significant differences were observed due to increased interanimal variability and relatively small tissue cpm values.
Linear regression analysis of intestinal loop retention of radioactivity (Fig. 2) was performed using the differences in tissue radioactivity calculated in formula (1). This analysis demonstrated an inverse correlation between concentration of unlabeled IGF-1 in the intestinal loops and the net difference of retention of radioactivity between paired pre-incubation and control segments (r = 0.70, P < 0.01). In addition, net retention of radioactivity in liver specimens was also significantly correlated with that of paired intestinal segments for both pre-incubation and control animals (r = 0.72, P < 0.01).
Characterization of Radioactivity
TCA precipitation of intestinal loop and liver homogenates was performed to estimate the portion of retained radioactivity in specimens that was intact peptide. TCA treatment of intestinal loop homogenates demonstrated that 49.7 ± 2.6% of the radioactivity present within the loop segments of controls was precipitated. No differences in TCA precipitation were noted for intestinal loops of the other (pre-incubation) groups and so results were pooled. Overall, 49.9 ± 1.7% of the radioactivity in intestinal segments was TCA precipitable. Similar findings were observed for liver TCA precipitable activity (40.3 ± 1.3%).
Selected intestinal loop homogenates were subjected to acid-gel chromatography to determine whether a significant portion of the radioactivity obtained in the samples co-migrated with native IGF-1. Figure 3 shows the results of a typical chromatograph of several pooled specimens. As depicted, approximately 40% of the radioactivity from intestinal loops exposed to 125I-IGF-I migrated in a position identical with intact IGF-1.
Milk-borne growth factors such as Epidermal Growth Factor and the Insulin-Like Growth Factors (18) have been implicated in growth and development of the intestine during early postnatal life. In addition, the use of enterally administered growth factors, either separately (19) or in combination (20), has been suggested as therapy for a variety of gastrointestinal disorders in later life. Recent reviews have also noted the potential of transgenically altered animals to overexpress specific growth factor genes exclusively in milk (21) or to develop altered forms of growth factors possessing enhanced biologic activity for enteral use (22).
In addition to the direct role that such growth factors might have on growth and development of the intestine, substantial evidence now exists that specific peptides such as IGF-1 may not only remain in receptor active form within the suckling intestine for as long as 30 minutes post ingestion (16), but may also be absorbed into the circulation in biologically relevant amounts (7). Some evidence has been presented that this process may also occur in the adult mouse (23). The mechanism(s) by which intestinal absorption of intact polypeptides might occur in animals is not well described, however.
Transcellular transport of IGF-1 has been a topic of research for a number of years. Bar et al. (24), using rat myocardium, have shown that this process occurs predominantly via modulation by tissue-specific IGF carrier proteins (IGF binding proteins (25)). Others (26) have suggested that transport of IGFs across vascular endothelial cells is at least in part modulated by IGF receptors expressed at the endothelial cell surface. However, Bastian et al. (14), using human umbilical vein endothelial cells in culture, have demonstrated significant transcellular IGF-1 transport in these cells that was not receptor mediated. In the latter study, movement of IGF across these vascular endothelial cells was not inhibited by the presence of excess IGF-1, nor by blocking the Type 1 IGF receptor.
More recently, the transport of IGF-1 has been studied (15) in epithelial cells, including those of lung, kidney, and gastrointestinal tract (IEC-6 cells). Although IGF-1 has been shown to bind to specific receptors on each of these cell lines, a general feature of all epithelial cell types studied is the demonstration of a nonsaturable diffusion of IGF-1 via a para or transcellular route. Unfortunately, no dose response curves have been generated for this study. Booth et al. (27), using a similar tissue culture model of intestinal epithelial cell growth, have shown that low doses of IGF-1 stimulated proliferation of cells but that the addition of insulin to the medium induced an inhibitory or static effect. Similar results related to IGF and other milk-borne growth factors have been obtained by others (28). These studies suggest the possibility of dose-related effects of IGF-1 on epithelial cells. Hypothetically, low-dose effects of IGF would be receptor mediated and might produce effects on cell proliferation, thus potentially inhibiting transcellular IGF transport in favor of “local” use, internalization, and degradation. Higher doses of IGF would theoretically have effects once all IGF receptors were occupied. Intestinal epithelial transport of IGF under these circumstances would, therefore, be receptor independent and the degree of transport would be purely related to local IGF concentration.
The present study examined the mechanism of transintestinal transport of IGF-1 in an in vivo model utilizing an isolated intestinal loop approach in suckling rats. Previous studies in our laboratory have demonstrated that milk-borne IGF-1 can be found intact in jejunum of suckling rats for 30 minutes post ingestion (16) and that receptor active IGF-1 may also be demonstrated in portal blood after enteral administration (7). The current study confirms in an in vivo isolated loop model the previous in vitro work of others that the process of retention and absorption of IGF-1 is not inhibited by peptide excess. It is also the first demonstration that IGF retention and uptake are increased when a 200-fold excess of peptide is present locally.
Because of the IGF dose and volume limitations for the jejunal preparations, it was impossible to obtain sufficient radioactivity from intestinal or liver samples to perform receptor competitive inhibition studies to document retained “native” structure of the IGF-1. However, in previously mentioned work (16), the radioactive dose given via stomach tube was 50 to 100X that of the present study, which allowed for enhanced isolation and purification of the administered IGF. In that study, TCA precipitation of radioactivity in intestinal homogenates correlated highly with the fraction of radioactivity recovered that co-migrated with native IGF-1. This material was also found to be receptor active IGF (16). The TCA precipitation and gel chromatography studies outlined in the present work strongly suggest that 40 to 50% of radioactivity recovered from intestinal and liver specimens was associated with bioactive or “intact” IGF-1. However, if a greater proportion of the retained tissue radioactivity were due to non-bioactive or partially degraded peptide, it could suggest that greater luminal concentrations of unlabeled IGF increased the overall cellular processing of milk-borne IGF, with less left for transcellular transport.
In examining the regulation of intestinal transport of macromolecules, intestinal “closure,” or the ability of intestinal epithelial cells to form tight junctions (18,29), is an important concept. Permeability of the immature gut is reported to be greater than in the mature gut (26,30,31), although paracellular flow of some macromolecules occurs irrespective of age. Evidence is available that at least some other milk-borne bioactive factors are absorbed from intestinal sites into the blood stream with consequent peripheral effects (18,26,31,32). Both paracellular and transcellular transport of bioactive factors are likely to be significant factors involved in milk-borne growth factor delivery to the suckling.
In summary, using an in vivo model of enteral uptake of IGF-1 in suckling rats, we have shown that the process of retention and uptake is nonsaturable up to 1 μg/ml of IGF-I, a concentration 200 fold in excess of that in colostrum (6,7). Our findings suggest that this uptake may also be mirrored by increasing amounts of exogenous IGF-1 in the livers of study animals. Although these studies were performed in sucklings, the results may have relevance for the treatment of gastrointestinal conditions in later life as well.
This work was supported by funds from the National Institutes of Health (NICHD) P01 HD26013, the University of California Davis Health System and the Armstrong-McDonald Foundation. Portions of this work were presented at the Annual Meeting of the Society for Pediatric Research, Boston, MA, May 2000.
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