Effect of Gi-proteins on Insulin Binding, Internalization and Recycling of Insulin Receptor in Bovine Aorta Endothelial Cell

Kwon, Hyuk Ho; Son, Hyun Shik; Lee, Jung Min; Ko, Seung Hyun; Hong, Ok Ki; Moon, Sung Dae; Chang, Sang Ah; Yoon, Kun Ho; Cha, Bong Yun; Lee, Kwang Woo; Son, Ho Young; Kang, Sung Koo

Abstract

BACKGROUND: Guanine nucleotide binding proteins (G-proteins) play important roles in the hormonal actions of many signal transduction systems. Possible roles for the Gi-protein in insulin action have been suggested. It is reported that Gi-protein is associated with insulin actions to a greater extent than Gs-protein. There are at least three different subtypes of Gi-proteins (Gi(alpha1), Gi(alpha2), and Gi(alpha3)), however, it is not certain which subtypes are associated with insulin receptors and their action.
METHODS
To investigate the effects of Gi-proteins on insulin action, the Gi-proteins were overexpressed in cultured bovine aortic endothelial cells (BAEC), using the DNA-polylysine-adenovirus complex transfection method. After incubating for 24 hours, the BAEC were treated with 200 ng/mL insulin to evaluate the insulin binding, receptor internalization and recycling.
RESULTS
The following results were found : 1) The binding of specific insulin bindings to the insulin receptors of endothelial cells were time-dependent, reaching their maximal levels in all cells after 30 minutes. The maximal specific bindings of the control, Gi(alpha1), Gi(alpha2), and Gi(alpha3) were 0.58+/-0.1, 0.54+/-0.08, 0.54+/-0.1, 0.53+/-0.09%, respectively. 2) The internalization of 125I-insulin, into endothelial cells, was assessed by the acid washing dissociation method, and occurred rapidly. There was a significant difference in the internalized radioactivity of the 125I-insulin in the overexpressed Gi(alpha2) protein group compared to the two groups. 3) The recycling of the insulin receptors in the three types of Gi-protein showed no significant difference between the three group.
CONCLUSION
In conclusion, the Gi(alpha2) protein may be associated with internalization of the insulin-insulin receptor complex, and appears to be important in both the action of insulin and the intracellular processing of insulin receptors.