Breast milk has non-nutritional protective effects on recipient infants. It has been speculated that bioactive substances present in human milk have important roles in protecting infants. However, the mechanisms by which such substances protect newborns are unclear. Therefore, we analyzed the growth-promoting activity of human milk and the intracellular signaling mechanism thereof using human fetal small intestinal (FHS 74 Int) cells. Epidermal growth factor (EGF) stimulated the proliferation of these cells. However, this stimulation was less effective than that of aqueous milk (5% vol/vol). The bioactivity of human milk was heat stable but protease sensitive. EGF receptor tyrosine kinase inhibitor did not repress the milk-induced growth-promoting effect on fetal small intestinal cells. Regarding the intracellular signaling pathway, the milk-induced cell proliferation pathway was tyrosine kinase dependent but was neither mitogen-activated protein (MAP) kinase nor phosphatidylinositol-3 (PI-3) kinase dependent. On the other hand, EGF-induced cell proliferation was tyrosine kinase, MAP kinase, and PI-3 kinase dependent. Rapid tyrosine phosphorylation of several intracellular proteins was detected after milk stimulation. Furthermore, the time course of phosphorylation induced by milk was different from that induced by EGF. The sizes of the proteins phosphorylated in response to milk were different from those of the Shc proteins phosphorylated in response to EGF. These results suggest that human milk induces fetal intestinal cell proliferation through a unique tyrosine kinase pathway different from the EGF receptor signaling pathway.

Glucose is the main energy substrate in the bovine ovary, and a sufficient supply of it is necessary to sustain the ovarian activity. Glucose cannot permeate the plasma membrane, and its uptake is mediated by a number of glucose transporters (GLUT). In the present study, we investigated the gene expression of GLUT1, 3 and 4 in the bovine follicle and corpus luteum (CL). Ovaries were obtained from Holstein × Japanese Black F1 heifers. Granulosa cells and theca interna layers were harvested from follicles classified into five categories by their physiologic status: follicular size (≥ 8.5 mm: dominant; < 8.5 mm: subordinate), ratio of estradiol (E₂) to progesterone in follicular fluid (≥ 1: E₂ active;<1: E₂ inactive), and stage of estrous cycle (luteal phase, follicular phase). CL were also classified by the stage of estrous cycle. Expression levels of GLUT1, 3 and 4 mRNA were quantified by a real-time PCR. The mRNA for GLUT1 and 3 were detected in the bovine follicle and CL at comparable levels to those in classic GLUT-expressing organs such as brain and heart. Much lower but appreciable levels of GLUT4 were also detected in these tissues. The gene expression of these GLUT showed tissue- and stage-specific patterns. Despite considerable differences in physiologic conditions, similar levels of GLUT1, 3 and 4 mRNA were expressed in subordinate follicles as well as dominant E₂-active follicles in both luteal and follicular phases, whereas a notable increase in the gene expression of these GLUT was observed in dominant E₂-inactive follicles undergoing the atretic process. In these follicles, highly significant negative correlations were observed between the concentrations of glucose in follicular fluid and the levels of GLUT1 and 3 mRNA in granulosa cells, implying that the local glucose environment affects glucose uptake of follicles. These results indicate that GLUT1 and 3 act as major transporters of glucose while GLUT4 may play a supporting role in the bovine follicle and CL.

Free radicals, hydroxyperoxides and H(2)O(2) are all known to damage cell components. This study was designed to compare the concentrations of hydroxyperoxide and free radical scavengers in the cardiac muscles of old rats in the hyper- or hypothyroid condition, to determine whether rates of peroxidation would differ with age, thyroid status, or both. Rats were rendered hyper- or hypothyroid by administration of l-thyroxine or methimazole for 4 weeks. Among the old rats, the lipid peroxide (LPO) concentrations, measured as thiobarbituric acid (TBA) reactants, were significantly greater in the hyperthyroid than in the euthyroid state and the LPO concentrations measured as TBA+Fe(3+) reactants, which may be precursors of LPO, were significantly greater in the hyperthyroid state, whereas in young rats, the LPO concentrations measured by TBA or TBA+Fe(3+) methods did not differ significantly in the hyperthyroid state. In the euthyroid state, the concentration of LPO measured as TBA+Fe(3+) reactants was significantly reduced with age. Xanthine oxidase (XOD) activity also was markedly increased with age, being more pronounced in the hyperthyroid than in the euthyroid state. The Mn and Cu/Zn superoxide dismutase activities were greater in the hyperthyroid than in the euthyroid state. Glutathione peroxidase activity decreased with age in the euthyroid and, particularly, in the hyperthyroid state. Catalase activity was not affected in the old rats. Concentrations of alpha-tocopherol in the old rats were high in the hyperthyroid state and low in the hypothyroid state, whereas the levels of beta- and gamma-tocopherols in these rats were unchanged in both conditions as compared with the euthyroid state findings. Data suggest that the site of free radical generation differs in older rats, with additional shifts in the location of intracellular lipid peroxidation being noted during hyperthyroidism. Thus, as rats age, the reduction of the free radical scavenger system and the increase in LPO and XOD activities might induce myocardial dysfunction.

The deterioration of glucose metabolism frequently observed in hyperthyroidism may be due in part to increased gluconeogenesis in the liver and glucose efflux through hepatocyte plasma membranes. Glucose transporter 2 (GLUT 2), a facilitative glucose transporter localized to the liver and pancreas, may play a role in this distorted glucose metabolism. We examined changes in the levels of GLUT 2 in livers from rats with l-thyroxine-induced hyperthyroidism or methimazole-induced hypothyroidism by using Western blotting to detect GLUT 2. An oral glucose tolerance test revealed an oxyhyperglycemic curve (impaired glucose tolerance) in hyperthyroid rats (n=7) and a flattened curve in hypothyroid rats (n=7). GLUT 2 levels in hepatocyte plasma membranes were significantly increased in hyperthyroid rats and were not decreased in hypothyroid rats compared with euthyroid rats. The same results were obtained with a densitometric assay. These findings suggest that changes in the liver GLUT 2 concentration may contribute to abnormal glucose metabolism in thyroid disorders.