It has been known for more than 20 years that ingestion of colostrum by newborn piglets leads to a marked increase in gastrointestinal weight, length, and protein and DNA content within the first 24 h of life (1). More recently, direct stimulation of gastrointestinal DNA (2) and protein synthesis (3) by colostrum and, to a lesser degree, mature milk has been demonstrated. In addition to enhanced protein synthesis within the intestine, piglets fed colostrum for 6 h had greater fractional protein synthesis rates in liver, kidney, spleen, and skeletal muscle than piglets fed mature milk, indicating that colostral factors may mediate growth of tissues outside of the gastrointestinal tract (3). It was hypothesized that nutrients and other nutrient-independent factors were responsible for the stimulation of protein synthesis (4). Among the nutrient-independent factors in porcine colostrum and milk are growth factors, which include insulin-like growth factor-I (IGF-I) (5). IGF-I is a 7.5-kDa peptide that is a potent mitogen both in vivo and in vitro (6). IGF-I milk has been proposed to play a role in neonatal intestinal development (7).
For milk-borne IGF-I to stimulate protein synthesis in tissues outside the gastrointestinal tract, it must survive digestion, be absorbed, and be transported to its target tissue. Data from several recent studies have indicated that IGF-I in milk may retain bioactivity within the gastrointestinal tracts of neonatal calves (8) and rats (9). In addition, Burrin et al. (4) demonstrated that plasma IGF-I concentrations increased linearly during 24 h in piglets fed colostrum, and that plasma IGF-I concentrations tended to be higher in piglets fed colostrum than mature milk or formula, which suggests indirectly that IGF-I in milk may be absorbed by the neonatal piglet. In contrast, ingestion by piglets of sow milk replacer supplemented with high physiological (500 μg/L) (10) to pharmacological (10,000 μg/L) (11) doses of IGF-I for 14 and 4 days, respectively, did not result in increased serum IGF-I concentrations, which suggests that oral IGF-I was contributing significantly to serum IGF-I. Therefore, the goal herein was to determine the degree to which milk-borne IGF-I is absorbed by the newborn piglet by orally administering 125I-rhIGF-I and serially sampling venous and arterial blood for 4 h after feeding.
MATERIALS AND METHODS
125I-rhIGF-I Preparation and Administration
Recombinant human IGF-I (rhIGF-I) (Genentech, South San Francisco, CA, U.S.A.) was iodinated to a specific activity of 200 mCi/mg. Briefly, 10 μg of rhIGF-I was incubated with 1.0 mCi of Na-125I (Amersham, Arlington Heights, IL, U.S.A.) in the presence of 3.5 mM chloramine-T (Sigma Chemical Co., St. Louis, MO, U.S.A.) in 0.2 M sodium phosphate buffer for 30 s. The reaction was quenched by the addition of 0.2 M sodium phosphate buffer containing 25 g/L bovine serum albumin (Sigma). Tracer was purified by gel filtration on a 0.7 × 30 cm Econo-column (Bio-Rad, Richmond, CA, U.S.A.) containing Sephadex G-50 (fine, Pharmacia, Piscataway, NJ, U.S.A.). The column was equilibrated with 0.05 mol/L Tris buffer, 5 g/L BSA, pH 7.4 before iodination. Specific binding of the tracer by an anti-human IGF-I antibody was 65%.
The night before the study, 125I-rhIGF-I (≈20 μCi) was added to a commercial sow milk replacer (Advance, Milk Specialities, Dundee, IL, U.S.A.). Advance is a nutritionally complete diet for piglets that is comprised of 25% protein, 13% fat, 48% lactose, and 6.5% ash and provides 721 kcal/L. Vitamins, minerals, and trace elements met National Research Council (NRC) requirements for piglets (12). The sow milk replacer was mixed thoroughly in a 50-ml sterile polypropylene tube and was stored at 4°C until ≈1 h before use, when it was allowed to come to room temperature. An aliquot of the formula was counted immediately after the addition of the tracer and just before feeding to rule out loss of isotope via nonspecific adherence to the plastic tube. We determined that the milk replacer was devoid of IGF-I or IGF binding proteins (IGFBP) (data not shown), which is consistent with previous reports for human infant formulas (13).
Animals and Surgical Procedures
The animal protocol was approved by the Laboratory Animal Care Advisory Committee of the University of Illinois, and animals were cared for in compliance with the NRC Guide for the Care and Use of Laboratory Animals (14). Newborn, colostrum-deprived piglets (n = 6) were obtained by cesarean section of crossbred sows on day 113 of gestation (mean gestation in the sow is 113-115 days). Within 2 h of delivery, 3.5-F polyvinyl chloride catheters (Sherwood Medical, St. Louis, MO, U.S.A.) were inserted into the umbilical artery and vein using the method of Odle et al. (15). Briefly, piglets were lightly sedated with 2% isoflurane (Anaquest, Madison, WI, U.S.A.). The abdomen was washed and an iodine disinfectant was applied to the surgical region. To numb the umbilicus, a total of 15 mg of lidocaine (Anthony Products, Arcadia, CA, U.S.A.) was injected subcutaneously into multiple sites surrounding the umbilical stump. The arterial catheter was inserted 22 cm into the dorsal aorta to a position near the heart. A second catheter was inserted into the umbilical vein and advanced 9 cm into the portal vein to a position near the liver. A radiograph showing the final positioning of each catheter is shown in Fig. 1. Catheters were secured with suture and elastic tape and were flushed with heparinized saline (10 IU heparin/ml in 0.9% NaCl) to maintain patency.
After recovery from the anesthesia (≈30 min), baseline blood samples (2 ml) were drawn from each catheter and piglets were administered ≈25 ml/kg body weight of formula containing the 125I-rhIGF-I by orogastric gavage. Blood samples (2 ml) were drawn from each catheter 15 min postfeeding and then at 30-min intervals for 240 min. Blood volume was maintained by injecting 2 ml of heparinized saline into the catheter after each sampling. Plasma was obtained by centrifugation at 10,000 g for 15 min at 4°C. At the termination of the experiment, the piglets were killed and the intestines were separated from the stomach and cecum/colon and were removed. Each intestine was laid out in five turns so that it formed six segments of equal length as described by Zijlstra et al. (16). The intestine was then divided into 13 segments of approximately equal length corresponding to the duodenum (segments 1 and 2), the jejunum (segments 3-9), and the ileum (segments 10-13). Each intestinal segment was flushed with 2.5 ml of ice-cold saline and the segment and saline flush were retained for further analysis.
Plasma IGF-I concentrations at baseline (t0) were measured by radioimmunoassay (RIA) (5). To dissociate the IGF-I from IGFBP, plasma samples (0.5 ml) were separated over a 0.9 × 100-cm column containing Sephadex G-50 (fine) in 0.2 mol/L formic acid (Pharmacia) (5). Fractions containing the IGF-I peptide (46-71 ml) were collected into 50-ml tubes containing 0.25 ml of RIA buffer (0.03 mol/L sodium phosphate, 0.25% bovine serum albumin (BSA), 0.02% sodium azide, pH 7.5) and were frozen and lyophilized (Flexi-Dry, FTS Systems, Stone Ridge, NY, U.S.A.). IGF-I recovery from the column is >90% (5). The lyophilized IGF peptide fractions were resolubilized in RIA buffer and diluted 1:4. IGF-I content was measured using 125I-rhIGF-I as radioligand and a polyclonal anti-human IGF-I antibody (1:10,000 final dilution) distributed through the National Hormone and Pituitary Program. After an overnight incubation, bound radioactivity was precipitated by centrifugation at 3,000 g for 30 min after the addition of 250 μl of 1% bovine IgG (Sigma) and 1.0 ml of 20% polyethylene glycol (Sigma). Samples were run in a single assay with an intraassay coefficient of variation of 3%.
Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis and Western Ligand Blot Analysis of Plasma GFBP
Molecular forms of plasma IGFBP were characterized by Western ligand blotting as previously described (5). Plasma samples (4 μl) were mixed with sample buffer (10% glycerol, 2% sodium dodecyl sulfate (SDS), and 0.625 mol/L Tris-HCl, pH 6.8) and proteins were separated through 4% stacking (0.1% SDS, 0.125 mol/L Tris-HCl, pH 6.8) and 12% running (0.1% SDS, 0.375 mol/L Tris/HCl, pH 8.8) polyacrylamide gels at 65 V, 4°C overnight (Hoefer Scientific Instruments, San Francisco, CA, U.S.A.). Proteins were transferred to nitrocellulose (0.45 μm, Micron Separations Inc., Westborough, MA, U.S.A.) at 200 mA for 1 h using a Buchler semidry blotting unit (Labconco Corp., Kansas City, MO, U.S.A.). Membranes were sequentially blocked with (Tris-buffered saline (TBS); 0.15 mol/L sodium chloride, 0.01 mol/L Tris HCl, pH = 7.5) containing 3% Tergitol NP-40 (Sigma). TBS containing 1% BSA (Sigma), and TBS containing 0.1% Tween (Sigma), and then were incubated overnight with 0.45 μCi of 125I-rhIGF-I. IGFBP were visualized by autoradiography (Eastman Kodak, Rochester, NY, U.S.A.) at -70°C for 4 days. Band density was quantified using the Foto/Analyst II Visionary System and Collage software (Fotodyne, New Berlin, WI, U.S.A.).
Total Acid-Precipitable, and Immunoreactive Counts in Plasma
The total radioactivity was determined in 0.5 ml of plasma by gamma counting (Cobra Gamma Counter, Packard Instruments, Downers Grove, IL, U.S.A.). To determine the proportion of acid-precipitable radioactivity, an equal volume (0.5 ml) of 25% trichloroacetic acid (TCA) was added to the sample plasma samples. After a 15-min incubation on ice, tubes were centrifuged at 10,000 g for 15 min, the supernatant was removed, and the amount of radioactivity in the pellet was measured. To determined the proportion of radioactivity that was immunoreactive 125I-rhIGF-I, 0.5 ml of plasma was subjected to acid chromatography to dissociate the IGF-I from the IGFBP as already described. Samples were lyophilized and resuspended in RIA buffer. Duplicate 50- and 100-μl aliquots were incubated overnight at 4°C with the hIGF-I antibody used in our IGF-I RIA at a 1:1,000 final dilution. Bound radioactivity was precipitated and radioactivity in the pellet was determined as already described. Immunoprecipitated radioactivity was interpreted as intact IGF-I. However, because we did not verify the size of the peptide that was immunoreactive, it is possible that some fragments of IGF-I were also detected. To achieve as complete an immunoprecipitation of intact 125I-rhIGF-I as possible given that ≈1.5-2.0 nmol/L of native IGF-I would be present in the serum samples, we conducted a pilot study in which porcine serum samples were spiked with a known amount of 125I-rhIGF-I and were incubated with increasing amounts of antibody. A final dilution of 1:1,000 in the assay buffer was adequate to overcome any competition by native IGF-I.
Each intact intestinal segment was weighed and its length was recorded. The radioactivity in a 3-4 cm sample from the middle of each intact segment was measured. Radioactivity within the intestinal mucosa was not measured separately from the underlying tissues. Radioactivity in a 0.5-ml aliquot of the intestinal saline flush was determined and the sample was TCA-precipitated as described for plasma.
Analyses were performed using the general linear modeling procedure within SAS (version 6.08, SAS Institute, Cary, NC, U.S.A.). Plasma was analyzed in a two-way analysis of variance (ANOVA) with time and blood (arterial versus portal) and the interaction between these factors included in the model. A one-way ANOVA was used for intestinal samples. Fisher's PLSD test was used if a main treatment effect was detected. Significance was assigned at the 0.05 level. All data are expressed as mean ± SD.
Dose of IGF-I Administered
Piglets (n = 6) weighed 1.25 ± 0.09 kg. Piglets received 24.2 ± 1.8 ml/kg body weight of formula. Mean 125I-rhIGF-I dose was 21.7 ± 1.8 μCi or 17.4 ± 0.7 μCi/kg body weight. Based on a specific activity of 125I-rhIGF-I of 200 mCi/mg, piglets received ≈13.1 nmol (100 ng) of IGF-I, which is comparable to 1 ml of porcine colostrum or 5-10 ml of mature porcine milk (5). Recovery of the tracer was determined at the end of the study (t240). Approximately 25 ± 2.8% of the administered dose was recovered, of which 8.0 ± 1.8% was present in the plasma at t240, 13.6 ± 2.7% was associated with the intestinal wall, and 2.9 ± 1.3% was recovered in the intestinal contents.
Total, Acid-Precipitable, and Immunoprecipitable Radioactivity
The time course of arterial and portal plasma total radioactivity is shown in Fig. 2. Radioactivity was detected in both portal and arterial plasma within 15 min of administration of 125I-IGF-I; however, radioactivity in the portal plasma exceeded that of arterial plasma at 15 min postgavage. After 30 min, no significant difference between the total radioactivity in portal and arterial samples was observed. Radioactivity peaked at 18.6 ± 3.5 μCi/L in portal plasma and 17.7 ± 3.9 μCi/L in arterial plasma at t240.
The percentages of total radioactivity that were TCA-precipitable and immunoprecipitable are summarized in Table 1. TCA-precipitable counts averaged between 18 and 20% of the total radioactivity in both arterial and portal plasma, and the proportion declined over time (p < 0.001). Of the total radioactivity in plasma, ≈3-5% was specifically immunoprecipitated by a polyclonal antibody to IGF-I (Table 1). Immunoprecipitable radioactivity was greater in portal than arterial plasma only at t15.
Plasma IGFBP Profiles
A representative Western ligand blot of arterial and portal plasma IGFBP profiles for one piglet is shown in Fig. 3, and densitometric analyses of the IGFBP profiles for all six piglets are summarized in Table 2. Four IGFBP bands were apparent at 43, 34, 28, and 24 kDa. The molecular weights of the IGFBP are comparable to those reported in neonatal piglets by McCusker et al. (17). The 43-kDa band most likely represents IGFBP-3, although usually two glycosylated variants of IGFBP-3 at 43- and 39-kDa are observed in older pigs (5,17). The 34-kDa band corresponds to IGFBP-2, which is the predominant IGFBP in neonatal pig plasma (17). The 28- and 24-kDa bands represent IGFBP-1 and -4, respectively. No differences in portal and arterial IGFBP profiles were observed. Only IGFBP-4 was significantly affected by feeding, being elevated in both portal and arterial plasma at 180 min postgavage.
Contribution of Absorbed IGF-I to the Circulating IGF-I Pool
Data used for calculating the contribution of 125I-IGF-I to the circulating IGF-I pool is summarized in Table 3. Plasma volume was estimated at 0.062 ± 0.005 L using a factor of 50 ml/kg body weight, which was determined for 20-kg immature pigs (18). It is possible that this value underestimates the plasma pool of the newborn. However, because the same plasma volume was used to determine the IGF-I and 125I-IGF-I pools, the relative contribution of the 125I-IGF-I to the total pool would be unaffected by using an alternate factor for determine plasma volume. Baseline plasma IGF-I concentrations were 1.81 ± 0.56 nmol/L, resulting in a circulating IGF-I pool of 0.113 ± 0.036 nmol. To estimate the potential contribution of milk-borne IGF-I, 125I-IGF-I immunoreactive counts at t240 were converted to nmol/L using a specific activity of 200 mCi/mg and the molecular weight of IGF-I (7.649). Based on these estimations, absorbed IGF-I from an oral dose of 100 ng would account for 0.025% of circulating pool of IGF-I of the newborn piglet. The t240 time point was chosen because the total radioactivity (Fig. 2) and the immunoprecipitable radioactivity had remained fairly stable for at least 1 h before sampling. It is likely that this value (0.025%) represents the minimal level of absorption, because clearance of the tracer from the circulation before t240 was not determined and colostral intake of IGF-I would be >100 ng.
Radioactivity in the Intestine and Lumenal Contents
The average weight and length of the small intestine were 32.9 ± 7.9 g and 255.2 ± 17.1 cm, respectively. The 125I recovered within the small intestine is shown in Table 4. Intestines were flushed with ice-cold saline to remove any 125I-rhIGF-I not adhering to the lining. Approximately 13.8% (3.0 ± 0.6 μCi) of the administered dose was associated with the intestinal wall. Radioactivity was lowest in the proximal duodenum (segment 1) and the distal ileum (segment 13). The remaining segments contained on average 0.2-0.3 μCi, or 7-10% of the recovered radioactivity. Only 0.63 ± 0.29 μCi, or 3% of the administered dose, was recovered in the lumenal contents (saline flush). There was no significant difference between the segments either for total radioactivity (data not shown) or when expressed as the percent recovered within each segment (Table 4). TCA-precipitable radioactivity within the lumenal contents of each segment averaged 15-30% of total radioactivity (data not shown); however, no significant differences were observed between the segments. No immunoreactive 125I-IGF-I was detected in the lumenal contents 4 h after oral administration (data not shown).
The absorption of milk-borne hormones and growth factors including prolactin and epidermal growth factor has been previously demonstrated, suggesting a potential role for these milk-borne factors as endocrine signals to the developing neonate (7,19). Indeed, the first hormone that was shown to be absorbed in an intact, bioactive form from milk was insulin (20), which shares significant structural and functional homology with IGF-I (6). Previous data from calves and piglets fed diets varying in IGF-I content suggested indirectly than milk-borne IGF-I may be absorbed. Neonatal calves fed colostrum had higher plasma IGF-I concentrations than calves fed mature milk (21) or a milk replacer devoid of IGF-I (8). Similarly, serum IGF-I concentrations tended to be higher in piglets fed colostrum than mature porcine milk or a commercial sow milk replacer, although the absolute concentrations did not differ between the groups (4).
Two studies in calves (8) and neonatal rats (9) have directly assessed absorption of milk-borne IGF-I by oral administration of 125I-IGF-I. Baumrucker et al. (8) administered 160 μCi of 125I-rhIGF-I to calves within 4 h of birth and collected blood samples for ≈22 h. Peak plasma radioactivity achieved in the calves (1.4-1.8 μCi/L) was 10-fold lower than was observed in the current study (14-18 μCi/L). A major difference between the studies was that in the calf study (8), 125I-rhIGF-I was added to 1 L of bovine colostrum that contained 45.8 nmol/L of native IGF-I, whereas in the current study 125I-rhIGF-I was added to a sow milk replacer that did not contain IGF-I. If IGF-I absorption occurs via a receptor-mediated pathway (22) or another saturable route, the IGF-I in the bovine colostrum could have competed with the 125I-rhIGF-I for absorption. In addition, bovine colostrum contains several IGFBP (23) that may affect IGF-I absorption, whereas the sow milk replacer used in our study was devoid of IGFBP. Despite these differences, a similar degree of absorption of intact IGF-I may have occurred. When plasma from the calves was pooled during the 1 st 5 h of the study, ≈12% of the pooled peak was immunoreactive (8), whereas in the current study ≈3-5% of the radioactivity was immunoprecipitable. Although it seems that both the newborn calf and piglet absorb a small percentage of orally administered IGF-I, it is likely to make only a negligible contribution to circulating IGF-I concentrations. In addition, extrapolation of these results to the human infant may not be appropriate. The degree of absorption in valves and piglets may be greater than in human infants because these species absorb colostral immunoglobulins by macromolecular absorption, and gut closure in piglets normally does not occur until 18-36 h postnatally (24). It has been previously demonstrated that colostrum increases the macromolecular absorption of markers (bovine serum albumin and fluorescein isothiocyanate-dextran) orally administered to newborn piglets (25). Therefore, it is possible that IGF-I uptake could have been enhanced if we had administered the 125I-rhIGF-I in colostrum rather than milk replacer.
Philipps et al. (9) orogastrically administered 4.5 μCi (0.9-1.3 nmol) of 125I-rhIGF-I or 125I-IGF-II to 10-11-day-postpartum rat pups. The 125I-rhIGF-I or 125I-rhIGF-II was mixed 1:1 with rat milk, which contained endogenous IGF-I, IGF-II, and IGFBP (26). Pups were killed 30 min after administration of the 125I-rhIGF-I, blood samples were obtained, and all tissues were removed. Approximately 42 and 38% of the administered radioactivity was associated with the gastrointestinal tissues (stomach wall and lumen and small-intestinal wall and lumen) in rats given 125I-rhIGF-I and 125I-rhIGF-II, respectively. For both peptides, <4% of the radioactivity was detected in blood and <2% in other organs including liver, kidney, brain, and lung. In the current study, 13% of the radioactivity was recovered in the small-intestinal lining and <3% in the small-intestinal lumenal contents, which may have been a result of differences in the timing of sampling (30 min versus 4 h postgavage). In addition, it is likely that tracer was cleared from the circulation before t240 and that tracer was associated with tissues that were not measured in the current study including the stomach, large intestine, and to a limited extent, tissues outside the gastrointestinal tract (9). We did not assess radioactivity within the gastric lumen or wall, because we had chosen a sampling time after which all formula had passed through the stomach (27). Urinary excretion of 125I was assessed in a pilot study in which piglets were fitted with bladder catheters in addition to the arterial and portal catheters. Urine samples collected during the 4-h study contained 1.1 μCi or ≈5% of the administered dose, of which none was immunoreactive (data not shown).
Using Sephadex G-50 gel filtration chromatography and high-performance liquid chromatography, Philipps et al. (9) found that ≈70% of the radioactivity in the stomach wall and 63% of the radioactivity in the gastric lumenal contents was intact 125I-rhIGF-I. Within the intestine, ≈50% of the recovered radioactivity in the small intestinal lumen and 10% in the small-intestinal wall was intact 125I-rhIGF-I. In addition, 125I-rhIGF-I recovered from the stomach wall bound to type I IGF receptors present in rat placental membrane, suggesting that orally administered IGF-I retains bioactivity for at least 30 min. In contrast, we were unable to detect any immunoreactive 125I-rhIGF-I in the small-intestinal lumenal contents 4 h postfeeding.
The ability of IGF-I to retain bioactivity for at least 30 min after oral administration supports several recent reports of intestinal effects of oral IGF-I in calves (28,29) and piglets (10,11). Baumrucker et al. (28,29) fed neonatal calves milk replacer or milk replacer containing 98 nmol/L of rhIGF-I for 7 days. Binding studies with intestinal cell microsomal membranes demonstrated that calves fed the milk replacer with added IGF-I had higher 125I-rhIGF-I binding capacity than calves fed milk replacer alone (28). In addition, [3H]thymidine incorporation into intestinal explants was greater per unit of DNA in calves receiving formula containing IGF-I (29). The authors speculated that upregulation of the type I receptor may be responsible for the enhanced thymidine incorporation in calves receiving IGF-I. In addition, several intestinal functional tests were performed in the calves (29). Using γ-glutamyl-transferase absorption, no effect of orally administered IGF-I on the degree of macromolecular absorption or the timing of gut closure was observed in calves (29).
Two recent studies have also demonstrated intestinal actions of oral rhIGF-I in the neonatal piglet. Colostrum-deprived piglets were fed sow milk replacer alone or supplemented with rhIGF-I at a dose of 200 μg kg-1 day-1(10) or 3,500 μg kg-1 day-1(11) for 14 or 4 days, respectively. Final body weight (10,11), organ weights (10), serum IGF-I (10,11) and IGF-II concentrations (10), and IGFBP profiles (10) were unaffected at either dose of rhIGF-I. That a pharmacological dose of 3,500 μg kg-BW-1 day-1 did not affect serum IGF-I concentrations supports the results of the current study, indicating that orally administered IGF-I is poorly absorbed. Small-intestinal weight, villus height, and intestinal protein and DNA content were significantly higher in piglets fed 3,500 μg rhIGF-I kg-1 day-1(11), but not in piglets fed 200 μg rhIGF-I kg-1 day-1(10). However, significant increases in villus height and digestive enzyme activity were apparent at the lower dose (10). Villus height in the distal ileum was 40-60% greater in piglets consuming 200 μg rhIGF-I kg-1 day-1 and the specific activities of sucrase in the jejunum and lactase in the jejunum and proximal ileum were two- to fourfold higher in piglets receiving rhIGF-I orally (10).
In conclusion, findings from other studies have demonstrated that orally administered IGF-I associates with the small-intestinal lining. In rats, orally administered IGF-I retains bioactivity within the intestinal wall for up to 30 min (9), which supports the ability of orally administered IGF-I to influence intestinal growth and development (10,11,28,29). Results of the current study indicate that although orally administered IGF-I exerts intestinal actions, it is poorly absorbed by the neonatal piglet and does not contribute significantly to circulating IGF-I concentrations, and therefore would have limited systemic effects.
Acknowledgment: This work was supported by funding from the National Institutes of Health (RO1 HD-29264).
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