Effect of Oxidezed LDL in Insulin Binding, Internalization and Recycling of Insulin Receptor in Cultured Bovine Aortic Endothelial Cells

Moon, Sung Dae; Cha, Bong Yun; Kim, Hye Soo; Jang, Sang Ah; An, Yu Bae; Song, Ki Ho; Han, Je Ho; You, Soon Jib; Yoon, Kun Ho; Kang, Moo Il; Lee, Kwang Woo; Son, Ho Young; Kang, Sung Koo

Abstract

BACKGROUND: Endothelial dysfunction is perhaps one of the earliest manifestations of atherosclerosis. This abnormality is in part due to altered membrane signal transduction in endothelial cells. Oxidized LDL that is atherogenic may induce endothelial dysfunction, and its presence has been documented in atherosclerotic vessels. Many studies have shown that oxidized LDL inhibits signaling pathways mediated by inhibitory GTP-binding proteins (Gi- protein). It is also known that G-protein is involved in insulin recycling on cultured human umbilical vein endothelial cells. Therefore, to determine the effect of oxidized LDL on endothelial cells: insulin binding, internalization, and the recycling of insulin receptors were assessed in cultured bovine aortic endothelial cells treated with native LDL, oxidized LDL, and in some cells pretreated with pertussis toxin before the incubation with oxidized LDL. METHOD: Native LDL (density 1.019 1.063 g/mL) was obtained from using the rapid single discontinuous density gradient ultracentrifugation of plasma samples from a single donor. Oxidized LDL was prepared by exposing samples of native LDL to CuSO4 (5 uM) at 37't for 24 hours. Endothelial cells at 80% confluence were treated with the indicated concentrations of native LDL, oxidized LDL, and some cells were pretreated with pertussis toxin for 6 hrs before the incubation with oxidized LDL. These cells were incubated for 24 72 hours.
RESULTS
1. Binding of (125)I-insulin(0.17nM) to endothelial cells treated with increasing concentrations of oxidized LDL shows dose-dependent decrease. There were significant differences in insulin binding between native LDL and oxidized LDL-treated cells (p<0.05). Binding of 'I-insulin (0.17 nM) to endothelial cells treated with increasing culture time of oxidized LDL shows more decreased than that of native LDL significantly (p<0.05). And oxidized LDL had additive effect, but not significant, with pertussis toxin on the specific (125)I-insulin binding to bovine aortic endothelial cells. 2. Internalization of insulin receptors reached rapidly to its maximal level around 30min at 37'C. At 60 min, oxidized-LDL treated cells was less increased in internalization of insulin receptors than that of native LDL treated cells [59.1+1.9% of total cell associated insulin (mean+SE) vs. 67.5+1.1%, p<0.05]. There were additive effects, but not significant differences, between oxidized LDL and pretreated with pertussis toxin before the incubation with oxidized LDL. 3. After 30 min of incubation with unlabeled insulin (33 nM), insulin binding in oxidized LDL treated cells was significantly higher compared to native LDL treated cells (69.0+2.5% of control values vs. 63.7+1.2%, p<0.05), suggesting that oxidized-LDL decreased internalization of insulin receptors. And during the process of recycling, there were significant differences in insulin receptor recycling between the oxidized LDL and native LDL treated cells, but oxidized LDL had an additive effect, but not significant, with pertussis toxin on insulin receptor recycling to the bovine aortic endothelial cells.
CONCLUSION
1. The findings in this study suggest that oxidized LDL may play a causative role to produce the insulin resistance by inhibiting insulin binding, internalization and recycling of insulin receptor in cultured bovine aortic endothelial cells 2. This study suggests that the effect of oxidized LDL to the bovine aortic endothelial cells in insulin binding and receptor-mediated transcytosis is caused by inhibiting pertussis toxin sensitive Gi-protein.