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M Sakata
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M Yamaguchi
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T Imai
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C Tadokoro
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Y Yoshimoto
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Y Oka
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H Kurachi
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A Miyake
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Abstract

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

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T Yoshimoto
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M Naruse
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Z Zeng
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T Nishikawa
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T Kasajima
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H Toma
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S Yamamori
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H Matsumoto
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A Tanabe
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K Naruse
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H Demura
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To explore the clinical significance of p53 in the pathogenesis of adrenal neoplasms, we investigated the incidence of p53 gene mutations in functioning human adrenal tumours using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique to screen p53 exons 4 to 9. We examined 29 adrenocortical adenomas (primary aldosteronism, n=17; Cushing's syndrome, n=12, all benign), and 33 phaeochromocytomas (benign solitary, n=18; benign multiple, n=5; malignant, n=10) in Japanese and Chinese patients. PCR-SSCP did not show any abnormal band-shifts in any of the adrenocortical adenoma and benign solitary phaeochromocytoma tissues. In contrast, six phaeochromocytoma tissues (two cases benign multiple, four cases malignant) showed PCR-SSCP band-shifts. Subsequent DNA sequencing analysis of the shifted bands revealed six cases with nine mutations or intronic sequence alterations: three cases contained sequence alterations within intronic regions, three cases with silent mutation (sequence alteration in codon without amino acid alteration), and three cases contained missense mutations (one case each in exons 5, 6 and 9). Immunohistochemical staining demonstrated that two of three cases with missense mutations and one case with an intronic sequence alteration over-expressed p53 protein in tumour cell nuclei. We observed no association between p53 gene mutation and p21/WAF1/Cip-1 expression. The relatively high incidence of p53 gene mutations or intronic sequence alteration in multiple and malignant phaeochromocytomas, but not in benign solitary cases, suggests that p53 mutation could play some role in the pathogenesis of multiple and/or malignant phaeochromocytomas.

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K Ogura
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M Sakata
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Y Okamoto
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Y Yasui
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C Tadokoro
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Y Yoshimoto
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M Yamaguchi
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H Kurachi
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T Maeda
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Y Murata
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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.

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