| Budget Amount *help |
¥3,400,000 (Direct Cost: ¥3,400,000)
Fiscal Year 2004: ¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 2003: ¥1,800,000 (Direct Cost: ¥1,800,000)
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| Research Abstract |
We have studied whether low-density lipoprotein(LDL) affected on the insulin secretion of pancreatic beta cells. We speculated that the modified LDLs affected on beta cell function after the uptake of LDL through lipoprotein receptor, so at first we checked that the lipoprotein receptors were found in the pancreatic beta cells. The immunostaining of the beta cells was performed with anti-rat insulin antibody, anti-LDL receptor(LDL-R) antibody, and anti-scavenger receptor antibodies (CD36 and SRB1). The positively stained cells with anti-insulin antibody were also stained with anti-LDL-R antibody, anti-CD36 antibody and anti-SRB1 antibody. These results indicated that the beta cells had not only LDL receptor, which was ubiquitously expressed, but also scavenger receptors concomitantly. In order to study how mRNA of each receptor was expressed in beta cells, we prepared the total RNA from rat liver, kidney, islets of pancreas and Hit-T15 cells, which were the cell line derived from hamst
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er pancreatic beta cells. The rat liver expressed each mRNA of LDL-R, SRB1, and CD36. The rat kidney had both receptors mRNA of LDL-R and SRB1, but not CD36 mRNA, which had already been reported in anywhere. In the rat islets and Hit-T15 cells each mRNA of LDL-R, SRB1 and CD36 was expressed as same as liver. Therefore it was suggested that lipoproteins would be up taken through the receptors and affect on insulin secretion. In order to check the lipoprotein uptake, Hit-T15 cells were cultured with DiI-labeled native LDL (nLDL), oxidized LDL (oxLDL), and acetylated LDL (AcLDL). Hit-T15 cells were clearly marked under the fluorescence microscopy, which suggested that every LDLs were internalized into the cells. These results suggested that each LDL was uptaken through each receptor. Next we checked the insulin secretion in Hit-T15 cells cultured with each LDL. OxLDL (25 or 50 μg/ml) were added into the culture of Hit-T15 cells for 24 or 48 hours. The glucose concentrations of the medium were set at 1,3,and 5mg/ml. The insulin secretion and the intracellular insulin contents in the culture with oxLDL were disturbed in the dose and time dependent manner. The expression of insulin gene was significantly decreased in the oxLDL addition. These results were not found in nLDL and AcLDL added culture. These changes of the insulin metabolism in Hit-T15 cells were caused by oxLDL and not by AcLDL. As conclusions oxLDL affected on the beta cells disturbing insulin gene espression and decreasing the intracellular insulin contents and the insulin seceretion. These have been published in Biochim Biophys A - Mol Cell Biol L(1687:173-180,2005). Less
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