Insulin-receptor interaction in the rat hepatoma H-35 cell line

Author

Lajwanti S. Khemlani

Date of Award

12-1989

Degree Type

Thesis

Degree Name

Master of Science

Major Professor

John W. Koontz

Committee Members

Wesley D. Wicks, Jorge Churchich

Abstract

Binding of insulin to the cell surface insulin receptor is the first step in insulin action. Upon binding, the insulin-receptor complex is internalized and the receptor depending on the cell line is either recycled or is degraded.

In KRC-7 cells, a subclone of the H-35 rat hepatoma cell line, insulin binding parameters such as the binding affinity (K_d) and the receptor number per cell have been determined for the cell surface, particulate cell membranes and solubilized receptor. Competition inhibition studies have been performed with control cells and cells incubated with a high concentration of insulin. Results from cell surface insulin binding to control cells have indicate a single, high affinity insulin binding site on the cell surface. Cells incubated with insulin demonstrate a 75% reduction in insulin binding. Scatchard analysis of this data indicate that the decrease in insulin binding is due to the decrease in cell surface receptor number per cell and not due to decrease in the binding affinity. To calculate the total receptor number insulin binding to the particulate cell membrane and solubilized receptor has been performed. And, Scatchard analysis indicate the possibility of the presence of an intracellular low affinity binding site. In KRC-7 cells insulin binding to the solulbilized receptor from cells incubated with insulin demonstrate that the internalized receptor does not remain functional in terms of insulin binding.

Insulin-receptor interaction studied in the presence of wheat germ agglutenin (WGA), a plant protein, has indicated that WGA increases insulin binding by two fold. Preliminary insulin binding in the presence of WGA performed in our laboratory has indicated the WGA increases the isnulin binding affinity and not the insulin receptor number. Insulin receptor aggregation has been postulated to be the reason for the increase in insulin binding affinity in the presence of WGA. Therefore, to determine whether WGA induces receptor aggregation, cross - linking studies have been performed in the presence and in the absence of WGA. SDS electrophoresis and autoradiography have been utilized to visualize insulin receptor subunits. Results indicate that WGA does not induce receptor aggregation in KRC-7 cells.

Recommended Citation

Khemlani, Lajwanti S., "Insulin-receptor interaction in the rat hepatoma H-35 cell line. " Master's Thesis, University of Tennessee, 1989.
https://trace.tennessee.edu/utk_gradthes/12986