Diabetes & Metabolism Journal

Original Articles

Effect of Oxidized LDL on the Amount of Insulin Receptor and Gi-proteins in the Caveolae of Bovine Aortic Endothelial Cells (BAEC).: Sung Yoon Jeon, Hyun Shik Son, Jung Min Lee, Sung Dae Moon, Kun Ho Yoon, Bong Yun Cha, Kwang Woo Lee, Ho Young Son, Sung Koo Kang; Korean Diabetes J. 2001;25(1):71-82. Published online February 1, 2001

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Abstract PDF: BACKGROUND
AND AIMS: Oxidized LDL (ox-LDL) may induce endothelial cell dysfunction and suggested to have an association with atherosclerosis or insulin resistance. Several studies have shown that ox-LDL inhibits signaling pathways mediated by inhibitory GTP-binding proteins (Gi-proteins). G-protein coupled receptors (GPCRs) can be internalized via caveolae. Caveolae are small flask-shaped invaginations of the plasma membrane, characterized by high levels of cholesterol and glycosphingolipids and also by the presence of caveolin, a 20-24 kDa integral membrane protein. G-proteins are enriched within caveolae membranes, where caveolin-1 directly interacts with the -subunits of G-proteins. It is reported that functional changes of G-proteins such as mutational or pharmacological activation of G-proteins affect direct interaction between G-proteins and caveolin-1. Thus, we investigated the effect of ox-LDL on the change of the amount of insulin receptor and Gi proteins in the caveolae. MATERIALS AND METHODS: ox-LDL was prepared by exposing samples of native LDL (n-LDL) to CuSO4 for 24 hours. Caveolae were extracted after treating BAECs at several concentrations of ox-LDL (10, 50, 100 g/mL) for various durations (0-48 hr), and we investigated the changes of the amount of caveolin-1, Gi -proteins and insulin receptor using immunoblot. RESULTS: While the amount of caveolin-1 was decreased, the amount of insulin receptor, Gi 2 and Gi 3 proteins in caveolae were also decreased after treatment of ox-LDL on the BAECs (insulin receptor: 66%; Gi 2 protein: 33%; Gi 3 protein: 66%, p<0.05). The amount of caveolin-1 was increased for the first 6 hours and then decreased, however, the amount of Gi -proteins and insulin receptor were vice versa during 48 hours incubation. CONCLUSION: These results indicate that ox-LDL can affect the change of the amount of insulin receptor and Gi-proteins in caveolae and it may induce endothelial cell dysfunction.

Effect of Oxidezed LDL in Insulin Binding, Internalization and Recycling of Insulin Receptor in Cultured Bovine Aortic Endothelial Cells.: Sung Dae Moon, Bong Yun Cha, Hye Soo Kim, Sang Ah Jang, Yu Bae An, Ki Ho Song, Je Ho Han, Soon Jib You, Kun Ho Yoon, Moo Il Kang, Kwang Woo Lee, Ho Young Son, Sung Koo Kang; Korean Diabetes J. 1999;23(3):243-255. Published online January 1, 2001

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Abstract PDF: 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.

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