R. K. Rao

Fate of Insulin-Like Growth Factors I and II Administered Orogastrically to Suckling Rats

ABSTRACT: Milk-borne insulin-like growth factors I and II (IGF-I and -II) may be of importance in the differentiation of the gastrointestinal tract of the suckling. To test this hypothesis, 10- to 11-d-old suckling rats were given via an orogastric tube 125I-IGF-I (n = 6) or 125I-IGF-II (n = 6) in rat milk and killed 30 min later. The results of this study demonstrated that approximately 40% of the radioactivity administered was detected in the gastrointestinal tract for both 125I-IGF-I and 125I-IGF-II experiments. Gel chromatography of acid extracts of homogenates of gastrointestinal tissues and luminal contents demonstrated that a significant fraction of recovered radioactivity eluted in a position identical to “native” IGF. These findings were confirmed by subjecting similarly treated samples to high performance liquid chromatography. In addition, radioactive material recovered from Mr 7,500 fractions bound specifically to crude membrane IGF-I and -II receptor preparations, further suggesting the preservation of biologic activity of the recovered peptides. Although skin homogenates contained large peptide fragments of 125I-IGF-I, no “intact” IGF was found in the blood or other tissues. These findings suggest that milk-borne IGFs are stable in the neonatal gastrointestinal tract and remain biologically active for as long as 30 min postingestion.

Presence of multiple forms of peptidase inhibitors in rat milk.

Summary Peptides such as somatostatin (SS14), epidermal growth factor (EGF), transforming growth factor-α (TGFα), and insulin-like growth factors (IGF-I and IGF-II) are present in breast milk from various species, and their significance in the developing gastrointestinal tract has been suggested. Our recent studies have indicated that rat milk soluble fraction (RMSF) protects SS14 in the gastrointestinal lumen by inhibiting in vitro the luminal peptidolysis. In the present studies, we have shown that RMSF inhibited in vitro degradation by midjejunal luminal flushings of suckling rats of 125I-labeled somatostatin 14[Tyr11], EGF, TGFα, IGF-I and IGF-II, as well as trypsin activity in vitro against benzoyl-L-arginyl-p-nitroanilide. The inhibitory factors present in the RMSF were further fractionated by gel filtration on Sephadex G100, ion-exchange chromatography on DEAE-Sephadex, and fast protein liquid chromatography (FPLC). Gel filtration of Sephadex G100 separated RMSF into three peaks of proteins: G1, G2, and G3; peptidase inhibitor activities were present exclusively in G1. Ion-exchange chromatography on DEAE-Sephadex column resolved peptidase inhibitory activity (G1) into three different peaks, D1, D2, and D3, eluted at sodium chloride concentrations of 0.05 M, 0.1 M, and 0.2 M, respectively. Further purification of D2 by FPLC resulted in a fraction rich in peptidase inhibitory activity, which was essentially free of trypsin inhibitory activity. Results indicate the presence of at least three peptidase inhibitors in rat milk, which may play a role in the protection of milk-borne peptides in the gastrointestinal lumen.

PROCESSING AND TRANSFER OF EPIDERMAL GROWTH FACTOR IN DEVELOPING RAT JEJUNUM AND ILEUM

Using everted sac technique we demonstrated the transfer of 125I-mEGF across the jejunal and ileal walls of suckling, weanling and adult rats. The transfer by the suckling rat jejunum and ileum was significantly inhibited by the presence of dinitrophenol and sodium azide or by the replacement of sodium with potassium or choline, RP-HPLC analysis detected carboxy-terminal processing of 125I-mEGF in suckling and adult rat jejunum and ileum. Suckling rat jejunum produced 125I-des(53)mEGF and 125I-des(49-53)mEGF, whereas 125I-des(48-53)mEGF was detected in suckling rat ileum or adult rat jejunum and ileum. All three forms of 125I-mEGF bound to anti-EGF antibody and EGF receptors. The receptor binding of 125I-des(53)mEGF was higher than that of 125I-mEGF, but those of 125I-des(49-53)mEGF and 125I-des(48-53)mEGF were greatly diminished. Results indicate a carboxy-terminal processing of mouse EGF during uptake and transfer in the small intestine of developing and adult rats, and the resulting products showed altered receptor binding. An identical amino acid sequence of the C-terminal pentapeptide of eGF from mouse, human and possibly rat may suggest a biological significance of C-terminal processing of EGF in the small intestine.

Appearance of exogenous epidermal growth factor in liver, bile, and intestinal lumen of suckling rats.

This study was performed to investigate the distribution and the degradation of IV administered [125I]rat epidermal growth factor (rEGF) in the liver and gastrointestinal tract of suckling rats. The bile duct of anesthetized rats was cannulated, and [125I]rEGF was injected (with or without 2500-fold excess unlabeled rEGF) into the femoral vein. After 5, 30, 60, and 120 minutes, the radioactivity in the liver, stomach, small intestine, blood, kidney, bile, and luminal contents of the stomach and small intestine was measured. The extracted radioactivity was then analyzed by immunoaffinity chromatography and binding to EGF-specific receptors. High levels of radioactivity were found in the liver (57% of total administered) and small intestine (10%) at 5 minutes, which gradually decreased. On the contrary, radioactivity secreted in the bile and luminal contents of the small intestine increased with time. The radioactivity in the bile represented 2.4% and 4.5% of the total administered at 60 and 120 minutes, respectively. During the first 60 minutes, more than 90% of the radioactivity in the liver, small intestine, bile, and intestinal contents was immunoreactive. Thirty-four to seventy percent of the radio-activity in the bile and liver and 20%-41% of radioactivity in the small intestinal wall and contents were capable of binding to EGF-specific receptors. Radioactivity detected in the liver, bile, small intestine, and intestinal contents was profoundly reduced by the coinjection excess of unlabeled EGF. These studies show that IV administered [125I]rEGF is rapidly taken up by the liver and the gastrointestinal tract and secreted into the bile and intestinal luminal contents of suckling rats in form(s) capable of binding to anti-EGF antibody and EGF-specific receptors. The uptake and secretion by the liver and the small intestine appear to be receptor mediated.

Luminal processing of epidermal growth factor in mouse gastrointestinal tract in vivo

To test the stability of epidermal growth factor (EGF) in the gastrointestinal lumen 125I-labeled EGF was administered to the lumen of isolated stomach, duodenum, jejunum, midjejunum, and ileum of anesthetized mice (14-day-old neonatals and 8-week-old adults). Radioactivity extracted from luminal contents and tissues of gastrointestinal segments was analyzed by binding to C18 isolation cartridges followed by reversed-phase high performance liquid chromatography (RP-HPLC). At 10 min, 74-97% of administered radioactivity was present in the isolated segments (luminal contents + segment tissue). Major portions (60-88%) of radioactivity recovered from luminal contents and segment tissues bound to C18 cartridges, except for lower values (40-54%) recorded in segment tissues of adult mice. RP-HPLC identified > 90% of C18-extracted radioactivity from gastric luminal contents of neonatal and adult mice as intact [125I]EGF (30-min retention time). In adult mice, 46-51% of radioactivity extracted from midjejunal luminal contents was identified as intact [125I]EGF, whereas only 3-5% was intact [125I]EGF in neonatal mice. On the contrary, in extracts of duodenal, jejunal, and ileal luminal contents, 14-30% of radioactivity was intact [125I]EGF in neonatal mice, whereas < 3% was intact [125I]EGF in adult mice. Considerable amounts of intact [125I]EGF were present in the adult mouse gastric tissue and neonatal mouse gastric and duodenal tissues. The remainder of C18-extracted radioactivity from different luminal contents and segment tissues eluted as three major C-terminally truncated EGF derivatives. These three [125]EGF derivatives, eluted with retention times of 35, 21, and 24 min, respectively, were identified as 125I-labeled EGF(1-52), EGF(1-48), and EGF(1-47).(ABSTRACT TRUNCATED AT 250 WORDS)

Presence of insulin-like growth factors and their binding proteins in rat milk

The insulin-like growth factors (IGF’s) are potent mitogens in a variety of mammalian cell types in vitro and in vivo (1–3). Because of the rapid growth rate during the perinatal period, recent interest has focussed on the IGF’s as possible modulators of fetal and neonatal somatic and organ growth. Significant concentrations of IGF-1 and IGF-2 have been observed in fetal blood in a variety of mammalian species (4–6). In addition, recent demonstration of active IGF synthesis using probes for IGF mRNA in fetal and neonatal tissues has led credence to this hypothesis. Active synthesis and expression of Type 1 and 2 IGF receptors has been documented for a wide variety of cell types in several animal species in fetal life as well (7–8). Interestingly, although the production of IGF receptors in developing gastrointestinal (GI) tissues is high (9), expression of IGF specific mRNA is low in these tissues in comparison to that found in other organs such as liver and brain (7, 17) Since, in the neonate, GI tissues represent a rapidly dividing and differentiating cell mass, the possibility exists that exogenously derived IGF’s might play a role in postnatal GI growth. IGF’s are also found in most biological fluids tightly bound to at least several molecular weight species of binding proteins (BP’s). Whether these BP’s act to carry the IGF’s to distant sites, fulfilling an endocrine function; act as a reservoir of IGF; or act to protect IGF from proteolytic degradation is unclear at present.

Fate of intraduodenally administered somatostatin in rats in vivo

Intraduodenally administered somatostatin-14 (a milk-borne peptide) has been shown to influence the pancreatic secretions in rats and dogs. To delineate the mechanism involved in the intraduodenal somatostatin-14, the fate of intraduodenal somatostatin-14 was investigated by administering [125I][Tyr11]somatostatin-14 ([125I][Tyr11]SS14) into the lumen of isolated duodenum in vivo of suckling and adult rats. At 2, 5, 10, and 30 min after administration, the radioactivity in the duodenal contents, duodenal wall, blood, liver, and kidney was measured, and the extracted radioactivity was analyzed for intact [125I][Tyr11]SS14 by reversed-phase high performance liquid chromatography. Radioactivity disappeared rapidly from the duodenal lumen with 50% of loss occurring at 2 min. No trace of intact [125I][Tyr11]SS14 was detected in any of the samples, except duodenal contents of suckling rats at 2 min, suggesting a rapid metabolism of SS14 in the duodenal lumen and a lack of duodenal absorption of intact SS14 in the rat.

The developing gastrointestinal tract and milk-borne epidermal growth factor.

In adult mammals, epidermal growth factor (EGF) is produced mainly in the submandibular and Brunner’s glands1–3. The production of EGF in suckling mammals is considered to be very low. Popliker et a1.4 reported absence of EGF mRNA in suckling mice; others described only very small amounts of EGF to be present in submandibular glands of suckling rodents1,5–7. EGF is known to play an important regulatory role in the mammals; many studies suggest that EGF is a trophic factor for various regions of the developing gastrointestinal tract8–17 and liver18,19.

Milk-Borne Peptide Growth Factors in Human and Bovine Milk

Publisher Summary This chapter reviews the presence of growth factors in human and bovine milk, and discusses the possible fate of milk-borne growth factors in the neonate, using as an example data obtained in studies on epidermal growth factor in the suckling mammal. Human and bovine milk, and milk of other species, contains many peptide hormones and hormone-like substances in considerable concentrations. The chapter reviews data about the effects of these peptides in the gastrointestinal tract of adults after parenteral administration. The gastrointestinal tract of suckling animals can be influenced both by parenteral and by gastrointestinal administration. Experiments performed in a laboratory demonstrate that milk-borne epidermal growth factor “survives” in the gastrointestinal tract of suckling rats and in the gastric content of preterm human neonates. These in vitro observations correlate with results obtained in vivo in rats. It is also shown that the epidermal growth factor (EGF) content of the gastrointestinal tract of suckling rats reacts quickly to the orogastric intake of EGF.

Hormones in Milk: Their Presence and Possible Physiological Significance

The presence of hormones in breast milk and their physiological significance was suggested by Schein (1895) and Mosse and Cathala (1898) during the last century and described five decades ago by Heim (1931a; 1931b) and Yaida (1929). In the last decade we witnessed an increased interest in this area. In our presentation we will review two aspects of the presence of hormones in breast milk, along with a brief discussion of clinical studies considering their functional significance for the suckling infant, as well as experiments exploring absorption of some milk hormones in laboratory animals.