Growth of artificially fed infant rats: effect of supplementation with insulin-like growth factor I.

Insulin-like growth factor I (IGF-I), a potent mitogenic peptide, is present in considerable quantities in most mammalian milks, but its importance for the neonate is unknown. To test the hypothesis that milk-borne IGF-I is an important factor in the regulation of neonatal growth, as well as that of the gastrointestinal tract, rat pups were fed a rat milk substitute (RMS) devoid of growth factors via gastrostomy. These animals were compared with those given RMS supplemented with recombinant human IGF-I added at a concentration of 500 ng/ml. Animals given RMS + IGF-I gained mere weight than controls, although skeletal growth as represented by elongation of the tail was no different. Animals fed RMS + IGF-I had increased brain and liver wet weights as well as increased liver and small intestine protein contents. Serum IGF-I concentrations in the IGF-I-supplemented group were more than twofold above RMS controls and were similar to dam-fed rat pups. Semiquantification of serum IGF-binding proteins (IGFBP) in these animals documented that in IGF-I-supplemented pups the amount of 38- to 40-kDa molecular mass IGFBP species was also greater than in RMS controls. The rate of migration of enterocytes from crypts in duodenum and proximal jejunum was greater in IGF-I-supplemented animals than in rats fed RMS alone. These studies suggest that milk-borne IGF-I is important in modulation of somatic and gastrointestinal tract growth in the neonatal rat.

Epidermal growth factor and transforming growth factor-alpha mRNA in rat small intestine: in situ hybridization study.

The expression of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) mRNA in the small intestine of suckling and adult rats was examined by in situ hybridization. EGF mRNA was found mainly in the intestinal crypts in adult rats. Adult rats also exhibited a considerably stronger signal for EGF mRNA in comparison to suckling rats, where the signal was very low or absent. In contrast to EGF, very strong expression of TGF-alpha mRNA was observed in the small intestine of both adult and suckling rats. These data suggest the differences between the expression of EGF and TGF-alpha in the developing small intestine.

Immunocytochemistry of albumin in hepatocytes after overnight starvation and during a diurnal cycle in young rats.

The indirect immunoperoxidase method was used to identify albumin in hepatocytes of young rats before and after periods of starvation and during a normal diurnal cycle. All liver cells in fed rats contained an abundance of albumin, whereas hepatocytes from overnight fasted animals showed minimal amounts of the protein. Hepatocytes in rats on the diurnal cycle generally contained more albumin during the light phase than in darkness. At the beginning of the dark phase, certain hepatocytes were low in albumin and they were located primarily around portal canals. Halfway through the dark period, these cells had increased in number and were located closer to terminal hepatic venules. Overnight starvation of young rats profoundly lowers hepatocyte albumin and the time of highest liver cell albumin content in the diurnal cycle of fed, young rats is during the first half of the light period.

Routine overnight starvation and immunocytochemistry of hepatocyte albumin content.

The indirect immunoperoxidase method was used to identify albumin in hepatocytes of rats before and after periods of starvation. All hepatocytes in fed rats contained a relatively large amount of nascent albumin. Overnight fasting reduced the number of hepatocytes with a large amount of albumin to primarily those surrounding terminal hepatic venules. These were estimated to be about 30% of the population. The other cells had only a slight amount of albumin. After 48 h of fasting all hepatocytes contained a low level of albumin.

Ultrastructural immunocytochemistry of nascent albumin topology: proposed cytosolic folding and membrane transit of the protein.

The relationship of nascent albumin and hepatocyte organelles was studied with the immunoperoxidase reaction in rats given various drugs to alter cellular albumin content. colchicine was used to increase intracellular albumin. Cycloheximide inhibited synthesis but allowed nascent albumin to remain with its ribosome of origin. Puromycin also inhibited synthesis but released albumin from its ribosome. There was no difference in the appearance of attached ribosomes in hepatocytes from saline-injected rats and those given colchicine or cycloheximide. In these cases, membranes of the endoplasmic reticulum were consistently decorated with ribosomes positive for the presence of albumin antigenicity on their cytosolic surface. The cisternal and cytosolic compartments were negative. The situation after puromycin was different. Here the membranes appeared to be denuded of ribosomes and reaction product, indicative of albumin, was present only on the lumenal surface. To determine whether puromycin had caused the release of ribosomes, sections from puromycin-treated cells were stained nonspecifically with uranyl acetate. This showed that the normal amount of ribosomes was still bound but that they could not be seen when a probe specific only for albumin was used. It appears that nascent albumin can associate with its ribosome within the cytosol. Also, apparently after albumin passes through the membrane of the rough endoplasmic reticulum, it remains attached to its lumenal surface. A model incorporating cytosolic folding of albumin followed by its entropic membrane transit is presented.

A random arrangement of albumin-containing hepatocytes seen with histo-immunologic methods. I. Verification of the artifact.

The cellular and subcellular localization of albumin in hepatocytes of adult male rats was established with immunofluorescence and immunoperoxidase techniques. Livers were fixed while either filled or devoid of blood. In some rats, prior treatment with cycloheximide was used to deplete the albumin content of hepatocytes. Immunofluorescence of blood-free livers from untreated rats showed that all hepatocytes contained albumin. However, using the peroxidase method, the amount of immunoprecipitate in cisternae of the rough endoplasmic reticulum was so slight that specific localization of albumin was impossible. Yet in all cases, a positive reaction for the presence of albumin was seen on ribosomes attached to the endoplasmic reticulum. In contrast, immunofluorescence of blood-filled livers from untreated rats and those previously injected with cycloheximide showed that only a few scattered hepatocytes were positive for albumin. In these cases, subcellular localization of albumin was obvious because the immunoprecipitate was found in heavy concentration, buy only in the cytosol compartment.

A random arrangement of albumin-containing hepatocytes seen with histo-immunologic methods. II. Conditions that produce the artifact.

The cellular immunolocalization of albumin in rat liver has been studied as a function of various physiological and physical conditions. Our observations show that the prime requisite for accurate immunolocalization of albumin and other hepatic-based proteins is the complete removal of blood and especially plasma from sinusoids and the perisinusoidal space of Disse prior to fixation. Fixation of blood-filled liver specimens results in the antifactual entrance of plasma constituents into hepatocytes. When the fixative used in formaldehyde, the artifactual uptake occurs primarily into hepatocytes that have a high glycogen content. Fixation of blood-filled liver with acetic acid-ethanol causes a massive influx of plasma into all hepatocytes. On the contrary, with blood-free liver, varying the type of fixative consistently demonstrates that all hepatocytes normally contain albumin, transferrin, and fibrinogen simultaneously. Increasing the time between cessation of blood flow and outright fixation by either withholding the fixative or by impeding its diffusion through the specimen causes a progressive loss of antigenicity of albumin. The same result ensues when specimens remain in contact with the fixative for an extended time.

Asialoprotein uptake by liver cells: immunofluorescence microscopy.

Uptake of asialoproteins by hepatocytes causes a change in the intracellular pattern of immunofluorescence. Control cells display a peripheral fluorescence which probably represents nascent proteins. Dark nonfluorescent areas, that presumably contain glycogen, are located around the nucleus. In contrast, liver cells from rats injected with asialoproteins display a pancytoplasmic fluorescence due to an influx of endocytotic vesicles.

DNA synthesis and cell proliferation in the simple liver acinus of 10 to 20-day-old rats: evidence for cell fusion.

Radioautography after 3H-thymidine injection, mitotic arrest by colchicine and camera lucida drawings were used to study DNA synthesis, mitosis, formation of binucleated cells and morphogenesis in the simple liver acinus of Rappaport in 10-20-day old rats. By ten days the arrangement of hepatic cell plates had already attained the adult configuration - irregular and thick in acinar zone 1 (periportal), straight and thin in acinar zone 3 (pericentral). The DNA synthetic index of parenchymal and bile duct cells slowly decreased during the observation period. Zonal labeling remained steady in the relationship: zone 1 greater than zone 2 greater than zone 3. Mitosis of parenchymal and bile duct cells reached a peak at 12 days, decreasing slowly thereafter. Mitosis also exhibited the relationship zone 1 greater than zone 2 greater than zone 3. The number of binucleated cells remained constant until after day 14 when it increased rapidly. Zonal distribution of binucleated cells was just the reverse of that for DNA synthesis and mitosis, that is zone 3 greater than zone 2 greater than zone 1. Radioautographic studies of binucleated cells labeled with 3H-thymidine indicated that a small percentage of them were formed by fusion of mononucleated cells. Conclusions are: (1) the 10-20-day old rat liver is expanding its cell population primarily in acinar zones 1 and 2 while overt differentiation is occurring in acinar zone 3, (2) ingestion of solid food around day 16 may be related to binucleated cell production due in part to altered portal venous blood changing the cellular microenvironments, (3) binucleated cells arise by suppression of cytokinesis, cell fusion and/or other non-mitotic routes.

Precursor-product relationship between intrahepatic albumin and plasma albumin.

Rats were injected with [(3)H]leucine, and at various times thereafter labelled albumin was isolated by electrophoresis from their livers and blood plasma. The specific radioactivity of each protein was determined by spectrophotometry and liquid-scintillation spectrometry. Intrahepatic albumin was shown to be identical with plasma albumin by its electrophoretic mobility and antigenicity. It was found that intrahepatic albumin was the direct precursor of plasma albumin. Comparison of their specific radioactivities showed that intrahepatic albumin attained a higher specific radioactivity before plasma albumin. When plasma albumin reached its maximum specific radioactivity, that of intrahepatic albumin had decreased to a similar value. Thereafter, the specific radioactivity of intrahepatic albumin remained lower than that of plasma albumin.