| Project/Area Number |
08457052
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Ehime University |
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Principal Investigator |
SHIMAZU Takashi Department of Medical Biochemistry, Ehime University School of Medicine, Professor, 医学部, 教授 (30090400)
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| Co-Investigator(Kenkyū-buntansha) |
YANO Hajime Department of Medical Biochemistry, Ehime University School of Medicine, Assista, 医学部, 助手 (00284414)
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| Project Period (FY) |
1996 – 1997
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| Project Status |
Completed (Fiscal Year 1997)
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| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1997: ¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 1996: ¥3,900,000 (Direct Cost: ¥3,900,000)
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| Keywords | Brown adipocytes / L6 myocyters / GLUT1 glucose transporter / Noradrenaline / beta_3 adrenergic agonist / GLUT4 glucose transporter / cyclic AMP / ATB-BMPA / グルコース輸送体 / GLUT1 / GST-融合蛋白質 / 初代培養褐色脂肪細胞 / 交感神経 / ノルエピネフリン |
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| Research Abstract |
We have shown that the sympathetic neurotransmitter, noradrenaline (NA), or the beta_3-adrenergic agonist (BRL37344) enhances glucose uptake into cultured brown adipocytes and L6 myocytes by a mechanism involving beta_3-adrenergic receptors and cAMP,without causing translocation of either GLUT1 or GLUT4 glucose transporters from an intracellular pool to the plasma membrane. In order to determine which isoform of GLUT is responsible for the NA-induced activation of glucose transport, we labelled the exofacial glucose binding sites of GLUT1 and GLUT4 with a membrane-impermeable, photoactive bismannose derivative, ATB- [ ^3H ]BMPA.In contrast to the action of insulin, NA did not increase the exofacial photoaffinity labelling of GLUT4. However, NA was shown to increase ATB- [ ^3H ]BMPA labelling of cell surface GLUT1, without an increase in the amount of immunoreactive GLUT1. These results demonstrate that NA stimulates glucose transport in brown adipocytes by activation of the cell surface GLUT1 through a cAMP-dependent mechanism.
We have further analyzed the mcchanism of selective activation of GLUT1 by NA,assuming that certain regulatory protein (s) may interact with GLUT1 at their cytoplasmic domain and inhibit their intrinsic activity. To isolate such cytosolic protein (s) , we made glutathione S-transferase (GST) -fusion protein corresponding to the C-terminus of GLUT1 (GST-G1C) , and immobilized it on CNBr-Sepharose beads to prepare the GST-G1C affinity column. When cytosol proteins from brown adipocytes were subjected to affinity chromatography on this column, a 33 kDa protein was detected to bind specifically with the GST-G1C.The recovery of the 33 kDa protein was higher from the cytosol proteins of NA-treated adipocytes than those from control and insulin-treated adipocytes. These results suggest that the 33 kDa protein can be dissociated by treatment with NA,thereby releasing GLUT1 from the presumed inhibitory interaction.
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