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Suckling rats exhibit age-dependent differences in epidermal growth factor (EGF) levels in several organs. The present studies evaluated the effects of two hormones known for their maturative effect on suckling rats, cortisone and tri-iodothyronine (T3), on immunoreactive EGF levels in specific organs. Suckling rats were administered cortisone (5 mg/100 g body weight per day) or T3 (50 μg/100 g body weight per day) on days 8, 9, 10 and 11 after birth, and killed on day 12. Submandibular glands, kidneys, pancreas, liver and gastrointestinal tract mucosa and lumen were assayed for immunoreactive EGF by a speciesspecific radioimmunoassay. Low levels of EGF in the submandibular glands were increased slightly by both T3 and cortisone treatment. Cortisone evoked a tenfold increase in EGF in the pancreas, but had no effect on levels in the kidney or liver. In contrast, T3 evoked a sixfold increase in the EGF level in the kidney, but had no effect on levels in the pancreas or liver. Hormonal administration had no effect on EGF levels in the stomach. Within the intestinal tract, cortisone had no effect on the luminal EGF content of the duodenum, jejunum or ileum, but caused a decrease in the midjejunum. T3 evoked a decrease in the luminal EGF content of the ileum. The effect of cortisone on mucosal EGF content varied between regions; an increase was seen in the duodenum with a decrease in the midjejunum and ileum. T3 administration resulted in a significant decrease in EGF only in the mucosa of the ileum. The EGF-degradative capacity of the luminal contents of the jejunum and ileum, as studied in vitro, were increased by treatment with both T3 and cortisone. However, no direct correlation was observed between alterations of the EGF content in the lumen or mucosa and the increased degradative capacity. We therefore conclude that modulation of EGF levels within the intestine cannot be explained exclusively by alterations in the degradation rates. The results indicate that both adrenal and thyroid glands may play an important role in the modulation of EGF levels in the developing rat. Journal of Endocrinology (1991) 131, 95–100

The relationship between obesity and the digestion of carbohydrates is poorly understood. Data in humans have provided conflicting results. Studies using the obese mouse (C57BL/6Jobob) suggest that obesity is associated with increased activity of intestinal alpha-disaccharidases. To evaluate the developmental pattern of these enzyme activities in obesity, we determined the activity of sucrase and lactase in the small intestine of genetically obese mice (C57BL/6Jobob) and lean littermates at 3 and 10 weeks of age. Sucrase and lactase activities were measured on intestinal homogenates from segments of the small intestine in mice maintained on standard laboratory diets during the postweaning period. Results were expressed as specific activity and total activity per intestinal segment. Obese mice did not differ from lean littermates in body weight at 3 weeks of age, but exhibited increased protein content in the proximal small intestine. Sucrase specific activity was significantly higher in the obese mice at 3 weeks of age in all intestinal segments. Sucrase total activity showed a similar pattern. At 10 weeks of age, body weights of obese mice were substantially greater than the lean littermates. Sucrase specific and total activities were also greater in the obese mice at 10 weeks of age. Lactase specific activity, however, was similar in both obese and lean mice at both ages studied. Lactase total activity was greater in the obese mice, consistent with their greater intestinal mass. These observations demonstrate that changes in the intestinal sucrase activity of the obese mouse precede the development of excessive body weight.

Transforming growth factor-alpha (TGF-alpha) is a biologically potent polypeptide detected in the gastrointestinal tract in suckling rats. The major goal of the present study was to test the hypothesis that the administration of TGF-alpha affects gastric emptying and small intestinal transit in suckling rats. Suckling (12-day-old) rats fasted 16 h received rat TGF-alpha subcutaneously (s.c.) or orogastrically in varying doses (0, 0.5, 1.0 microg/rat in 0.1% BSA). Control animals received 0.1% BSA only. Poly R-478 dye was used as a motility marker. Rats were decapitated 45 min after marker administration and the amount of dye in the stomach and the small intestine was measured by spectrophotometry. Subcutaneous administration of TGF-alpha significantly delayed stomach evacuation. In controls, the stomach contained 21.4 +/- 1.4% (mean +/- s(x)) of the Poly R-478 marker, whereas in TGF-alpha treated rats the stomach contained 37.2 +/- 2.8% of the total Poly R-478 dye given to animals. The delaying effect of TGF-alpha was time- and dose-dependent. Small intestinal transit was also significantly delayed. The proximal jejunum of TGF-alpha treated rats contained a 1.4-fold higher amount of marker in comparison with control rats. Orogastrically administered rTGF-alpha did not affect gastric emptying or intestinal transit. In conclusion, s.c. administration of rat TGF-alpha significantly delayed the gastrointestinal motility in vivo in suckling rats.

During our studies on gastrointestinal motility in suckling rats using 51Cr or 51Cr-EDTA as markers, we noticed that these markers--in contrast to studies in adult rats--"adhered" to the gastrointestinal wall of sucklings. We therefore decided to test the use of another non-absorbable marker Poly R-478 (an acetylated anthrapyridone chromophore linked to an polyamino-ethylene-sodium ethylene sulfonate copolymer backbone developed by the Dynapol Corporation (Palo Alto, CA). This new method has appeared to be useful.

Effects of early neonatal interventions on metabolic parameters later in life (s.c. late effects) were studied in rats using two models; namely, (a) the effects of premature weaning and (b) the effects of "dietary" manipulations during the suckling period (s.c. small vs. large litters). (a) Premature weaning of rats caused an earlier degeneration of spermiogenesis and elevated plasma cholesterol levels in adult animals when compared to levels found in animals weaned 12 days later (on day 30 after birth). In adult rats, radioiodine uptake in thyroid glands was lower in the group weaned prematurely. Premature weaning was followed by a decrease of corticosterone production in adrenal glands in adult animals; in female adult prematurely weaned rats, an elevated response of adrenal cortex to stressors was observed. Several other studies explored the "immediate" effects of early, premature weaning. (b) Early exposure to high fat diet evoked a hypercholesterolaemic response in adulthood following brief exposure to HF diet. Rats from litters reduced to 3 or 4 pups per mother on postnatal day 3 exhibited 2 days later plasma levels of cholesterol higher than in rats raised in large litters of 8 or 14. The difference between small and large litters was preserved for the whole lifespan of the animals. In adulthood, rats from small litters were fatter and had higher levels of plasma cholesterol and insulin. Other studies suggester that early dietary experience may regulate the pattern of drug metabolism in adult life. An inhibition of diurnal plasma corticosterone variation was found in rats overfed during the neonatal period and an increased stimulation of lipolysis by norepinephrine and lipogenesis by insulin was demonstrated in neonatally underfed rats. Interesting studies were reported in longitudinally studies in children: at the age of 9-12 year breast-fed children (for more than 6 months) had the highest cholesterol levels; on the other hand significantly increased levels of APO B, Apo A1, ATH index and Apo/B Apo A1 quotient (p0.05) were found in the nonbreast-fed group (27 references).

Early studies suggested endocrine type mother-pup interaction: 131I administered to suckling rats appeared via the urine of the suckling and mother's milk in the circulation of litter mates who were not injected with iodine; levels of thyroxin in rat milk were influenced by the status of the thyroid gland of the lactating rat. Administration of TRH (thyrotropin releasing hormone) to lactating mothers led to an appearance of unaltered hormones in the milk and stomach content of sucklings. TSH (thyroid stimulating hormone) or ACTH (adrenocorticotropic hormone) when given orogastrically to suckling rats increased thyroid hormones and corticosterone serum levels in suckling rats. Functional effects of gastrointestinal administration of insulin, bombesin (mammalian analog of gastrin-releasing peptide) and epidermal growth factor (EGF) are reviewed in detail (32 references).

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.

Milk from lactating Sprague-Dawley rats was assayed for epidermal growth factor (EGF)-like activities. A homologous radioimmunoassay (RIA) indicated the presence of immunoreactive material that competed in a nonparallel fashion with submandibular gland rat EGF (sm-r-EGF). The activity in the milk was extracted using antibodies to sm-r-EGF covalently linked to acrylamide beads. This activity was characterized by RIA, nondenaturing polyacrylamide gel electrophoresis, enzymatic digestion, radioreceptor assay, and ability to stimulate incorporation of [3H]thymidine into cultured fibroblasts. The presence of three distinct immunoreactive forms of EGF in rat milk were detected that competed with 125I-labeled r-EGF for binding to the EGF receptor and stimulated DNA synthesis in growth-arrested fibroblasts. Two of the forms are converted to the sm-r-EGF species by tryptic digestion as determined by RIA and migration rates in a nondenaturing polyacrylamide gel. The biological activities are stable to heating in 0.1 M acetic acid and also are stable at pH 9.0. Levels of r-EGF equivalents in milk were low at time of birth (6.3 +/- 1.7 ng/ml) and rose to 35.4 +/- 14.6 by days 4 to 6.

As shown previously, during degradation of sucrase-isomaltase in rat jejunum, degradation of the sucrase active site occurs before that of isomaltase active site [Goda & Koldovský (1985) Biochem. J. 229, 751-758]. To characterize further the process of sucrase-isomaltase degradation in jejunum, we determined the amounts of immunoreactive sucrase-isomaltase in rat jejunum by using a monoclonal-antibody-based enzyme-linked immunosorbent assay. By employing two alternative monoclonal antibodies (one reacting with the sucrase subunit and the other reacting with the isomaltase subunit), the amount of antigen-containing sucrase subunit and the amount of antigen-containing isomaltase subunit were separately quantified. In both upper and lower jejunum of rats, the amount of antigen-containing isomaltase subunit was always higher than the amount of antigen-containing sucrase subunit. This difference was attributable mainly to a degradation product of sucrase-isomaltase, which was identified as isomaltase monomer. Occlusion of pancreatic ducts for 18 h eliminated the difference between the amount of antigen-containing sucrase subunit and the amount of antigen-containing isomaltase subunit in both upper and lower jejunum. In jejunum of control animals, the molar ratio of sucrase subunit to isomaltase subunit was estimated to be 0.32-0.52, indicating that quite a large proportion of sucrase-isomaltase (48-68%) is present as degradation products (e.g. isomaltase monomer). These results support the model of degradation process of sucrase-isomaltase in brush-border membranes of rat jejunum, whereby degradation of sucrase subunit by the action of pancreatic proteinase(s) precedes degradation of isomaltase subunit.

Male rats were weaned normally (NW; day 30 after birth) or prematurely (PW; day 18) to a Purina Chow diet. Serum cholesterol levels and the activities of some enzymes of fatty acid and glucose metabolism were determined when the animals were 6 and 10 months old and, in the older group, also after 2 days of starvation. Blood cholesterol levels rose with age and at 10 months were higher in PW than NW rats. This difference disappeared after starvation. Hepatic pyruvate kinase (PK) activity was the same in fed NW and PW animals but was significantly higher in starved PW than NW rats. Hepatic phosphoenolpyruvate carboxykinase (PEPcK) activity was lower in NW than in PW rats, but this difference disappeared on starvation. In white fat, starvation caused a fall in PEPcK activity in both groups. In general, the effect of starvation did not accentuate the differences between the two groups. However, PEPcK activity in white fat increased with age about fourfold.

Two aspects of the physiology of the developing gastrointestinal tract are discussed. The first one deals with functional peculiarities of the developing gastrointestinal tract. Examples are shown that less active function in the suckling (eg, low pepsin digestion in the newborn stomach) may actually give the suckling an advantage for another process. The existence of pH close to neutrality in the suckling's stomach enables "survival" of the milk and lingual lipases that are then "cooperating" with low pancreatic lipase known to be present in the sucklings. The other aspect discussed deals with the role of various factors involved in postnatal maturation of the gastrointestinal function. Using sucrase activity as an example, the important role and mutual interaction of fetal (genetic?) programming, dietary changes, and hormonal maturation are reviewed.

Changes of activity of intestinal acid β-galactosidase, β-glucuronidase, N-acetyl-β-glucosaminidase and arylsulphatase were studied in suckling rats treated with cortisone (5mg/100g body wt. daily, started on day 9 postnatally) and compared with changes in control animals. Specific activities were not changed within the first 72h, but all enzymes decreased similarly 96h after the first injection. Total activities per ileum and animal were not changed within the first 48h, but within 72h a significant decrease was observed. Calculation of the rate of decrease of the hydrolases studied in cortisone-treated animals shows that it proceeds faster than the rate of renewal of enterocytes in this period.

1. 1. The fatty acid composition of triglycerides and phospholipids isolated from the lungs, adipose tissue, intestine and liver was determined in newborn rats, in rats aged 1, 10 and 30 days, and in adult animals. 2. 2. Only the myristic and lauric acid contents of the triglycerides were found to change with changes in their content in the diet (milk against the solid diet for adult animals). Marked changes occurred in the fatty acid composition of the triglycerides during development in the different organs but these could not always be related to changes in the composition of the diet. 3. 3. The composition of fatty acids in the phospholipids differed with age and between organs. The lungs had the most specialized phospholipids, containing the aldehydes of stearic and palmitic acids and differing from the other organs also in their content of the other acids. Changes in the fatty acid composition of the diet were not reflected in the fatty acid composition of the phospholipids, although pronounced developmental changes were observed.

1. The characteristics of acid and neutral beta-galactosidases isolated chromatographically from homogenates of the mucosa of the jejunum and ileum of suckling rats were studied. 2. The minimal molecular weight of the acid beta-galactosidase, as estimated by gel filtration on Sephadex G-200, was in the range 83000-105000, whereas for the neutral beta-galactosidase the estimated molecular weight was in the range 360000-510000. 3. The acid and neutral beta-galactosidases were inhibited competitively by galactono-(1-->4)-lactone, with respective K(i) values of 0.15mm and 1.1mm. Only the acid beta-galactosidase was inhibited competitively by sodium galactonate (K(i) 0.17mm). 4. Heat inactivation of both beta-galactosidases occurred according to first-order kinetics. The neutral enzyme was more labile, but bovine serum albumin protected acid enzyme only. 5. Urea treatment inactivated both beta-galactosidases, the neutral beta-galactosidase being more sensitive than the acid beta-galactosidase. 6. No differences were found between preparations from the jejunum and ileum.

Adult rats that had been fed on a low-starch high-fat diet for 7 days were force-fed with either the same diet or isoenergetic diets containing 40% of energy as either sucrose or lactose. Within 12h, the increase in jejunal lactase activity in sucrose- and lactose-fed rats was accompanied by a corresponding increase in immunoreactive lactase protein.

1. beta-Galactosidase activity was studied in homogenates and isolated microvilli fraction of jejunal mucosa from 14-day-old suckling rats. o-Nitrophenyl beta-d-galactoside served as the substrate. 2. The microvilli fraction contains about one-third of the total activity of the original homogenate. 3. The pH optimum of the beta-galactosidase was 3.5 in the total homogenate and supernatant fraction, whereas in the microvilli fraction the maximum activity was at pH5.5. 4. This work gives further support to the view that two beta-galactosidases exist in the jejunal mucosa.

Summary Active transport of glucose has been demonstrated in everted sacs of the jejunum and ileum from fetuses 11 to 19 weeks old.

Suckling (12- to 14-day-old) and weanling (30-day-old) rats were sacrificed 2 h after oral administration of 3H-labeled prostaglandin F2 alpha. Although radioactivity recovered from the stomach and small intestine (including contents) was slightly higher in sucklings (28.3 +/- 3.7%; n = 10) than in weanling rats (21.3 +/- 5.3%; n = 7), the liver of sucklings contained significantly higher amounts of counts (11.0 +/- 1.1 vs. 3.3 +/- 0.5%). Combined column and thin-layer chromatography of liver extracts showed more authentic prostaglandin F2 alpha in sucklings (11.0 +/- 0.5% of the liver counts) than in weanlings (7.0 +/- 1.1%). The liver of suckling rats contained a higher percentage of more polar metabolites (43.3 +/- 1.6 vs. 34.3 +/- 3.0%). These studies demonstrate differences in processing of oral prostaglandin F2 alpha in the early postnatal period.

The first part of the paper deals with the effect of changes in the mother's hormonal status on the development of the small intestine of her offspring. Administration of cortisone or triiodothyronine (T3) to pregnant rats evokes a precocious appearance of sucrase activity in the fetal small intestine. Administered to lactating rats, T3 evokes a precocious increase in sucrase activity in sucklings and leads to increased T3 levels and decreased TSH levels in the mother's milk and in sera of mothers and sucklings. These experiments show that (a) sucrase activity can be induced in the fetal period; (b) changes in the mother's hormonal balance during the fetal and suckling period can influence the development of the small intestine; and (c) levels of thyroid hormones in milk can be altered experimentally. The second part deals with jejuno-ileal gradients in villus size and sucrase activity. Both gradients appear in fetal jejunal and ileal implants developing in adult hosts. Our experiments indicate that (a) the gradients are already 'programmed' during the fetal period; and (b) direct contact with food, and proximity of the jejunum to the flow of digesta from the stomach, are not decisive for expression of the jejuno-ileal gradients, but may play a 'tuning' role.

Acid lipase was identified in the rat small intestine by using esters of 4-methylumbelliferone as substrates. Maximum activity towards the oleate ester was found at pH 4.0. In adult animals, the activity of acid lipase exhibited both latency and sedimentability, indicating a lyosomal localization. The activity of acid lipase was practically the same along the height of the villus, thus paralleling the distribution of acid beta-galactosidase. In adult rats, the activity of acid lipase in proximal (jejunum) and middle (mid-jejunum) sections of the small intestine was practically the same and exceeded the activity in the distal (ileum) section by a factor of 2. In suckling rats, the activity of the enzyme in the mid-jejunum exceeded that in the jejunum and ileum by 2.5- and 1.5-fold respectively. During postnatal development, the acid lipase activity of the mid-jejunum showed a peak between days 10 and 15, at which time it exceeded the adult mid-jejunum activity by 5–6-fold.

The short-term effects of high-protein/low-carbohydrate diet on aminopeptidase N activity were studied in the brush-border membranes of proximal jejunum and proximal ileum of adult rats. The animals were starved overnight and re-fed for 15 h either with a standard diet (20% protein, 55% carbohydrate, in terms of energy content) or with a high-protein/low-carbohydrate diet of equal energy content (70% protein, 5% carbohydrate). All rats consumed similar amounts of diet, and measurements were made 15 h after initiation of re-feeding. In the proximal jejunum a slight increase in aminopeptidase activity was observed after the high-protein intake. In contrast, considerable stimulation (52%) of the enzyme specific activity was obtained in the proximal ileum. This increase in ileal aminopeptidase activity was more prominent in the mature cells of the upper villus. To determine if the increase of aminopeptidase activity was due to an increased amount of enzyme protein, rocket immunoelectrophoresis was performed with detergent-solubilized brush-border protein from ileum on agarose gels containing anti-(rat brush-border) antiserum. When the same amount of enzyme activity was loaded on the gels, the peaks of immunoprecipitate for aminopeptidase were similar for animals fed on a standard or a high-protein diet. When the same amount of protein was loaded, the peak of immunoprecipitate for aminopeptidase was higher (81%) after a high-protein diet. These results showed that the high protein intake evoked an increase in aminopeptidase activity, with a concomitant increase in the amount of immunoreactive protein.

Milk-borne epidermal growth factor (EGF) survives in the gastrointestinal tract and is delivered in active form to peripheral organs. The dose dependence of EGF effects in sucklings needs to be clarified, as do possible interactions with other biologically active peptides in milk.

Colostrum and milk contain insulin, epidermal growth factor and prostaglandins. Experiments performed in our laboratory have demonstrated that these three substances can be absorbed by the gastrointestinal tract of suckling rats in biologically active form in considerable amounts. Prostaglandins are metabolized by the gastrointestinal tract during postnatal development. There are quantitative and qualitative differences in the metabolism of orally administered prostaglandins between suckling and weanling rats. Nevertheless, a significant amount of intact prostaglandin appears in the liver of suckling rats after oral administration. Gastric administered insulin can result in hypoglycemia. In suckling rats, there is a considerable decrease in blood glucose levels, while no effect is seen in 30-day-old rats. Epidermal growth factor is processed in the gastrointestinal tract as shown by size exclusion and affinity columns and surface receptor binding. Oral administration of epidermal growth factor has several effects on gastrointestinal functions. Analysis of epidermal growth factor processing by the gastrointestinal tract demonstrated postnatal developmental differences (either during normal development, or one accelerated by steroid treatment). These studies show that the gastrointestinal tract can be influenced by orally administered hormones known to be present in milk, and that these substances can be delivered to peripheral organs of suckling animals intact.