The Effect of Nitric Oxide on Insulin Binding and Insulin Receptor Recycling in Bovine Aortic Endothelial Cells

Kwon, Hyuk Sang; Hong, Oak Kee; Kim, Hee Soo; Lee, Jung Min; Kim, Sung Rae; Moon, Sung Dae; Jang, Sang Ah; Son, Hyun Shik; Yoon, Kun Ho; Kang, Moo Il; Cha, Bong Yun; Lee, Kwang Woo; Son, Ho Young; Kang, Sung Koo

Abstract

BACKGROUND: The coexistence of insulin resistance and endothelial dysfunction is commonly observed in a variety of metabolic and cardiovascular disorders, including athero-sclerosis and type 2 diabetes mellitus. Because nitric oxide (NO), or nitric oxide synthase (NOS), has been suggested as a significant contributing factor in the development of endothelial dysfunction and insulin resistance, reactive NO or NOS were investigated to see if they contribute to the insulin internalization pathway.
METHODS
The production of NO (Nitrite), the expression of eNOS (endothelial NOS), insulin binding and the insulin receptor internalization and recycling, following 48 hours of incubation with bradykinin (BK), acetylcholine (Ach), NG-monomethyl- L-arginine (L-NMMA) and N-nitro-L-arginine methylester (L-NAME) in Bovine aortic endothelial cells (BAECs), were examined.
RESULTS
The results were as follows: 1. In relation to the time course, the production of eNOS was increased, but was decreased after 8 hours of incubation. The production of eNOS in the L-NMMA and L-NAME treated groups was significantly decreased compared with that of the controls (p<0.05). 2. The specific insulin bindings to the receptors of the endothelial cells were maximized within 20 mins, and then decreased. At 20 mins, the binding rate of the L-NMMA treated group was significantly decreased compared to that of the controls. At a concentration of 0.4ng/ml of unlabelled insulin, the specific insulin binding of the L-NMMA treated group was significantly decreased compared to that of the controls (p<0.05). 3. The internalization of 125I-insulin into the endothelial cells, as assessed by the acid washing dissociation method, occurred rapidly. The internalized radioactivity of 125I-insulin, at 20 mins, was significantly increased in the BK and Ach groups compared with the controls (p<0.05). 4. The recycling of the internalized insulin receptors showed no significant differences between the study groups, but the recycling was slightly delayed compared with controls in the Ach group.
CONCLUSION
In conclusion, the NO generating substances, BK and Ach, and the inhibitory substance, L-NMMA, may influence the binding and internalization of insulin-insulin receptors. Our results suggest that NO might contribute to the transcytosis of insulin in BAECs