| Project/Area Number |
09671054
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
内分泌・代謝学
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
OGAWA Yoshihiro Kyoto University Graduate School of Medicine.Assistant Professor, 医学研究科, 助手 (70291424)
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| Co-Investigator(Kenkyū-buntansha) |
HOSODA Kiminori Kyoto University Graduate School of Environmental studies.Assistant Professor, 人間環境学研究科, 助手 (40271598)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Leptin / Transcriptional regulation / adipocytes / BeWo cells / Transfection / C / EBP alpha / Sp1 / 脂肪組織 / ゲルシフトアッセイ / プロモーター活性 / 肥満遺伝子 / 胎盤 / ルシフェラーゼ |
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| Research Abstract |
We examined the promoter activity of the human leptin 5'-flanking sequences in primary cultures of rat mature adipocytes and BeWo cells, a human trophoblast cell line. Various length of the human leptin 5'-flanking sequences were subcloned into the promoterless firefly luciferase expression vector. In rat mature adipocytes, the longest promoter (-2080 to + 108) construct showed a high-level transcription activity. When deletion proceeded from -2080 to -136, no significant changes in promoter activity were noted. This 136-bp region contained three Sp1 binding sites and a C/EBP binding site. When the Sp1 sites and C/EBP site were simultaneously mutated, the promoter activity was reduced to that of the promoterless construct. In BeWo cells, the longest promoter (-2080 to +108) construct also showed a high-level transcription activity. Deletion from -2080 to -1885 made no significant changes. However, the promoter activity was decreased by more than 80%, when sequences between -1885 and -1830 were deleted. Electrophoretic mobility shift assays revealed the presence of nuclear protein(s) binding to the sequences (-1885 to -1830) in BeWo cells but not in rat mature adipocytes. We also investigated the effect of protein kinases A and C on leptin production in adipocytes and BeWo cells. Leptin production was decreased by forskolin but unchanged by PMA.Leptin production in BeWo cells was increased by forskolin. The forskolin-induced increase in leptin production was completely suppressed by H89, an inhibitor of protein kinase A.Leptin production in BeWo cells was increased by phorbol myristate acetate (PMA). The PMA-induced increase in leptin production was completely suppressed by H7 and staurosporine, both of which are inhibitors of protein kinase C.The present study demonstrates that the human leptin gene transcription is differentially regulated between adipocytes and placental trophoblasts.
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