Studies of the mechanism of insulin resistance in hypertensive rats
Insulin resistance in skeletal muscle is of major pathogenic importance in several common human disorders including diabetes, hypertension and obesity, but the underlying mechanisms are unknown. In order to define mechanisms of insulin resistance, studies have been undertaken in skeletal muscle of the SHRSP, an animal model of hypertension, which exhibits insulin resistance in skeletal muscle and adipose tissue, when compared to the normotensive WKY control animal. Anti-peptide antibodies, directed against the phosphorylated or unphosphorylated residue Ser-1177 of human eNOS, were prepared and characterised, to allow the study of eNOS regulation in SHRSP skeletal muscle by phosphorylation at this residue. However, some doubt exists over the specificity of these antibodies and future studies have not been undertaken at this stage. Flotillin is a protein known to be involved in a P13-kinase independent pathway, which is required for GLUT 4 translocation and increased glucose uptake in response to insulin. Flotillin expression is increased in skeletal muscle from SHRSP and for this reason a yeast two-hybrid screen was undertaken using flotillin-1 as bait, the aim being to identify flotillin interacting proteins which have possible roles in insulin-stimulated glucose uptake. A number of interesting putative flotillin interacting proteins was identified in this screen, however one must be cautious as no duplicate clones were identified. Also, due to time constraints no biochemical studies have been undertaken to determine if the proteins identified do indeed bind flotillin. Studies of the 'classical' P13-kinase-dependent insulin-signalling pathway were also undertaken. It was demonstrated that key proteins involved in the insulin-signalling pathway (GLUT-4, IRS-1, IRS-2 and the P13-kinase p85 subunit) are expressed at similar levels and have similar subcellular distribution (on crude fractionation of skeletal muscle) in skeletal muscle of the SHRSP and the control WKY. Furthermore, levels and activity of PKB (a protein kinase downstream of P13-kinase) were similar in WKY and SHSRP, suggesting that the insulin-signalling pathway leading to activation of PKB in SHRSP skeletal muscle is intact.