Facilitative glucose transporter-1 (GLUT1) is expressed abundantly and has an important role in glucose transfer in placentas. However, little is known about the regulation of GLUT1 expression in placental cells. We studied the changes in placental GLUT1 levels in relation to changes in glucose concentration in vitro and in vivo. In in vitro experiments, dispersed mouse placental cells were incubated under control (5.5 mM) and moderately high (22 mM) glucose concentrations, and 2-deoxyglucose uptake into cells was studied on days 1-5 of culture. After 4 days of incubation under both conditions, GLUT1 mRNA and proten levels were examined by Northern and immunoblot analyses. Treatment of cells with 22 mM glucose resulted in a significant decrease in 2-deoxyglucose uptake compared with control, from day 2 to day 5 of culture. Moreover, GLUT1 mRNA and protein levels on day 4 of culture were significantly reduced in cells incubated with 22 mM glucose compared with control. Next, we rendered mice diabetic by administering 200 micrograms/g body weight streptozotocin (STZ) on day 8 of pregnancy. Animals were killed on day 12 of pregnancy and placental tissues were obtained. [3H]Cytochalasin B binding study was carried out to assess total GLUTs, and GLUT1 mRNA and protein were measured as above. [3H]Cytochalasin B binding sites in placentas from STZ-treated mice were significantly less than those in control mice. Northern and immunoblot analyses revealed a significant decrease in GLUT1 mRNA and protein levels in diabetic mice compared with the controls. These findings suggest that the glucose concentration may regulate the expression of placental GLUT1.
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K Ogura, M Sakata, M Yamaguchi, H Kurachi, and Y Murata
M Sakata, M Yamaguchi, T Imai, C Tadokoro, Y Yoshimoto, Y Oka, H Kurachi, and A Miyake
Glucose plays an important role in fetal development and energy metabolism. Facilitative glucose transporter-1 (GLUT1) has been found in placenta. However, little is known about GLUT1 modulation in placental cells. To examine changes in mouse placental GLUT1 levels caused by 8-bromo-cAMP, we performed 2-deoxyglucose uptake experiments, Northern blot analysis and immunoblot analysis using a primary mouse placental cell culture. Immunohistochemical analysis showed that GLUT1 was localized to the ectoplacental cone and the labyrinth zone of mouse placentas on days 7 and 11 of pregnancy respectively. Treatment of mouse placental cells with 250 μmol/l 8-bromo-cAMP resulted in a significant (P<0·01) decrease in glucose uptake on days 2–5 of culture. The inhibitory effect of 8-bromo-cAMP on glucose uptake was concentration-dependent. Glucose uptake was also inhibited by 100 μg/l cholera toxin and by 0·1 mmol/l forskolin. Northern blot and immunoblot analysis revealed that both GLUT1 mRNA and protein levels were also decreased by 8-bromo-cAMP. These findings suggest that 8-bromo-cAMP inhibits glucose transport activity in mouse placental cells in culture.
Journal of Endocrinology (1996) 150, 319–327
M Yamaguchi, K Tasaka, K Ogura, M Sakata, J Mizuki, and A Miyake
The regulation of mouse placental lactogen (mPL)-I and mPL-II secretion by activin and inhibin and the expression of activin and inhibin subunit mRNAs in the mouse decidua were examined. Activin-A at a concentration of 10 nm/l significantly inhibited mPL-II secretion by placental cells from days 9 and 12 of pregnancy. However, activin-A did not affect mPL-I secretion by cells from days 7 and 9 of pregnancy nor mPL-II secretion by cells from day 7 of pregnancy. By contrast, 10 nm/l inhibin activated mPL-II secretion by cells from day 12 of pregnancy. These effects of activin and inhibin on mPL-II secretion were dose-dependent. Follistatin, which binds to activin and blocks its bioactivity, completely eliminated the inhibitory effect of activin on mPL-II secretion. Incubation of placental cells from day 12 of pregnancy with activin-A resulted in a significant reduction of the mPL-II mRNA level assessed by Northern blot analysis. Northern blot analysis using poly(A)+ RNA extracted from the decidua indicated that mouse decidua, as well as the placenta, express all activin and inhibin subunits and that their gene expressions increased during gestation. The expression of these mRNAs in the decidua was much higher than those in the placenta. These findings suggest that activin and inhibin regulate mPL-II secretion and suggest the presence of an autocrine or paracrine regulation of mPL-II secretion in mouse placenta by activin and inhibin after mid-pregnancy in vivo.
Journal of Endocrinology (1995) 146, 469–474
M Yamaguchi, M Sakata, K Ogura, K Adachi, A Mammoto, and A Miyake
The effects of interleukin (IL)-1 and granulocytemacrophage colony stimulating factor (GM-CSF), which are present in the mouse placenta, on the secretion of mouse placental lactogen (mPL)-1 and mPL-II by placental cells were tested in vitro. IL-lα and IL-1β, 2·5 nmol/l each, significantly inhibited mPL-II secretion by cells from days 9 and 12 of pregnancy, but did not affect mPL-II secretion by cells from day 7 of pregnancy or mPL-I secretion by cells from days 7, 9 or 12 of pregnancy. GM-CSF had no effect on mPL-I and mPL-II secretion by cells from days 7, 9 or 12 of pregnancy. The inhibitory effects of IL-1α and IL-1β on mPL-II secretion were completely eliminated by the addition of antibodies to IL-1α and IL-1β respectively. Western blot analysis for mPL-II indicated that IL-1α significantly reduced the intensity of the mPL-II band. Steady-state levels of mPL-II mRNA, assessed by Northern blot analysis, were reduced by incubation of placental cells from day 12 of pregnancy with 2·5 nmol/l IL-1α for 5 days. Co-incubation of 0·25 pmol/l IL-1α, 25 pmol/l IL-6, and 25 pmol/l tumor necrosis factor-α, each of which did not significantly inhibit mPL-II secretion by itself, together inhibited mPL-II secretion. These results suggest that IL-1, but not GM-CSF, is a potent inhibitor of mPL-II secretion after mid-pregnancy, and that the combined action of cytokines can inhibit mPL-II secretion.
Journal of Endocrinology (1995) 147, 423–429
T Takeda, M Sakata, R Minekawa, T Yamamoto, M Hayashi, K Tasaka, and Y Murata
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.
I Sakata, T Tanaka, M Matsubara, M Yamazaki, S Tani, Y Hayashi, K Kangawa, and T Sakai
Ghrelin was recently isolated from the rat stomach as an endogenous ligand for the GH secretagogue receptor. Although it is well known that a large amount of ghrelin is produced in the gastrointestinal tract, developmental changes in ghrelin mRNA expression and differentiation of ghrelin-immunopositive (ghrelin-ip) and mRNA-expressing (ghrelin-ex) cells in the stomach have not been elucidated. In this study, we therefore investigated the changes in ghrelin mRNA expression levels and in the numbers of ghrelin-ip and -ex cells in the stomachs of 1- to 8-week-old male and female rats by Northern blot analysis, immunohistochemistry and in situ hybridization. Northern blot analysis showed that the level of weak ghrelin mRNA expression was low in the postnatal period but then increased in a dimorphic pattern, i.e. transient stagnation at 4 weeks in the male rats and at 5 weeks in the female rats. The number of ghrelin-ip and ghrelin-ex cells also increased after birth, and more numerous ghrelin cells were found in female rats than in male rats, and this finding was confirmed by Northern blot analysis. Ghrelin-ip and -ex cells first appeared in the glandular base of the fundic gland and then they were found in the glandular base and the glandular neck at 3 weeks of age, suggesting that the distribution of ghrelin cells is extended from the glandular base to the glandular neck during the postneonatal development period. This is the first report on detailed changes in postneonatal ghrelin expression level and in the number of ghrelin cells in the rat stomach. The sexual dimorphism of ghrelin expression and ghrelin cell differentiation suggest that ghrelin plays an important physiological role in the stomach.
H Adachi, H Kurachi, H Homma, K Adachi, T Imai, M Sakata, Y Matsuzawa, and A Miyake
Aged mice exhibit an increase in their body weight (BW), which is associated with fat deposit increase. Epidermal growth factor (EGF) concentration in the submandibular gland also increases with aging. We examined the effects of elevated EGF on the adiposity of aged female mice. Studies were started in two groups of animals consisting of sham-operated (n=10) and sialoadenectomized (n=10, Sx; surgical removal of the submandibular glands) mice at 8 weeks of age. Body weight gain and food intake were measured throughout 78 weeks of age in these two groups. Body weight was significantly less in the Sx group throughout 78 weeks, while food intake was not changed by Sx after 12 weeks of age. To examine further if EGF plays a role in the induction of adiposity in aged female mice, sham-operated animals were given 100 μl anti-EGF rabbit antiserum (anti-EGF group, n=5) or normal rabbit serum (control group, n=5) every 3 days, and Sx animals were given 5 μg/day EGF (Sx+EGF group, n=5) or saline (Sx group, n=5) from 78 weeks of age for 3 weeks. At 81 weeks of age, all animals of these four groups were killed, and carcass fat deposition and fat cell sizes were measured. Although the relative weights (weight ratio to BW) of the liver and kidney were not changed by Sx and anti-EGF treatment, the relative weights of mesenteric and subcutaneous fat tissues and adipocyte weights were significantly decreased in Sx and anti-EGF groups compared with the control group. Moreover, both acyl-CoA synthetase (ACS) and lipoprotein lipase (LPL) mRNA levels were significantly decreased by Sx or anti-EGF administration in mesenteric and subcutaneous fat tissues. On the other hand, EGF administration to Sx animals had no effect on BW, fat tissues and adipocyte weights, and ACS and LPL mRNA levels. The results, however, were consistent with the fact that adipose tissue EGF receptors were down regulated in Sx mice. These findings suggest that EGF may play a role in the induction of adiposity in aged female mice.
Journal of Endocrinology (1995) 146, 381–393
K Ogura, M Sakata, Y Okamoto, Y Yasui, C Tadokoro, Y Yoshimoto, M Yamaguchi, H Kurachi, T Maeda, and Y Murata
Facilitative glucose transporter-1 (GLUT1) is abundant in trophoblast cells and is responsible for glucose transport in the placenta. However, the change in GLUT expression in human placenta upon trophoblast differentiation remains to be clarified. Therefore, we first examined the localization of GLUT1 and GLUT3 using human first-trimester chorionic villi. We found that GLUT1 and GLUT3 were mainly localized to syncytiotrophoblast and cytotrophoblast cells respectively. We analyzed whether placental GLUT1 and GLUT3 expression changes during differentiation using a human choriocarcinoma (BeWo) cell line which is known to show functional and morphological differentiation in response to cAMP in culture. Treatment of BeWo cells with 8-bromo-cyclicAMP (8-bromo-cAMP) increased the level of hCG secretion and induced cell fusion leading to the formation of large syncytia. Treatment of BeWo cells with 8-bromo-cAMP also resulted in a significant increase in glucose uptake on days 2-3 of culture. The stimulating effect of 8-bromo-cAMP on glucose uptake was concentration dependent. Northern and immunoblot analyses revealed that the levels of mRNA and protein of GLUT1, but not of GLUT3, were significantly increased by 8-bromo-cAMP. These findings suggest that 8-bromo-cAMP stimulates GLUT1 expression with differentiation in BeWo cells.
K Adachi, H Kurachi, H Adachi, T Imai, M Sakata, H Homma, O Higashiguchi, T Yamamoto, and A Miyake
We studied the expression of epidermal growth factor (EGF) receptor protein and messenger RNA (mRNA) in human fallopian tubes at three stages of the menstrual cycle: early follicular (n=3), late follicular (n=3) and luteal (n=3). Immunohistochemical studies in the ampullary portion of the tubes showed that specific staining was localized to the epithelium and the vascular endothelium. Staining of the epithelium was intense at the late follicular and luteal stages, while it was weak at the early follicular stage. 125I-EGF binding study in the tubal plasma membranes revealed a class of high-affinity EGF receptors. Although dissociation constants were similar between the stages, numbers of binding sites at the late follicular and luteal stages were significantly (P<0·01) greater than those at the early follicular stage. Western blotting showed that tubal plasma membranes contain M r 170 000 EGF receptor protein. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage. Reverse transcription and polymerase chain reaction (RT-PCR) revealed that EGF receptor mRNA was expressed in all the 9 RNA samples (n=3 for each stage) from the tubal ampullary portion. The amounts were significantly (P<0·01, n=3) greater at the late follicular and luteal stages than those at the early follicular stage (by a competitive PCR). Increase in the amounts of EGF receptor protein and mRNA occurred in association with an increase in serum oestradiol but not progesterone levels. Next we examined whether EGF receptor and its ligands (EGF and transforming growth factor a) are directly induced by oestrogen. We found that specific staining for EGF receptor and its ligands in the tubal epithelium was detected (by immunohistochemistry) in postmenopausal women with oestrogen replacement (n = 3), but not in subjects without oestrogen replacement (n=3). These results suggested that EGF receptors in the human tubal epithelium are expressed in relation to specific stages of the menstrual cycle and that the expression may be induced by oestrogen.
Journal of Endocrinology (1995) 147, 553–563