The significance of plasma membrane cholesterol content on insulin mediated effects, specifically in key downstream events of insulin signalling have been well studied. However, there is a lack of understanding of how cell membrane cholesterol influences binding of insulin to its receptor.
In this study, the cell membrane cholesterol content of CHO T10 cells, a cell line overexpressing the insulin receptor, was either decreased or increased by treatment with methyl-β-cyclodextrin (MBCD) or cMBCD (cholesterol loaded MBCD) respectively. Similar studies were also performed on a hepatocyte cell line (HepG2) and human skeletal muscle myoblasts (HSMM). Influences on insulin binding and signalling were studied using competitive radio-ligand binding assays and Western blot analysis respectively.
Treatment of CHO T10 cells with 10 mM MBCD or cMBCD for 30 min resulted in a 0.7 fold decrease (P<0.0001) or 0.7 fold increase (P< 0.0001) respectively in cell cholesterol content compared to controls, with both treatments associated with an approximate 0.5 fold decrease (P < 0.0001) in insulin binding. Similar effects were observed in HepG2 and HSMM cells. While there was a decrease in binding, indicative of decreased receptor number, this was not associated with a change in receptor affinity in any cell type. Western blot analyses did however demonstrate a decrease in insulin signalling events in MBCD and cMBCD treated cells; an approximate one fold decrease (P < 0.0001) in phosphorylation of GSK3β and Akt was observed for both treatments relative to control.
Taken together, these data indicate that plasma membrane cholesterol influences cell surface insulin receptor content and signalling.