Mutated Insulin Receptor Val⁹⁹⁶ Reduces Insulin-Dependent Generation of Inositol Glycan and Diacylglycerol

We evaluated whether insulin-receptor tyrosine kinase activity is required for activation of PDH, insulin-induced hydrolysis of PIG and generation of IG and 1,2-DAG. For the analysis, we used stable-transfected CHO cell lines expressing wild-type human insulin receptor (CHO-wt cells) or the mutant receptor (Val⁹⁹⁶) that lacks tyrosine kinase activity (CHO-mut cells) (1,2). Insulin stimulated PDH activity in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to activate PDH was 7 × 10⁻¹¹ M in the CHO-wt cells, 10⁻⁹ M in the parental cells, and 8 × 10⁻⁹ M in the CHO-mut cells. Insulin stimulated hydrolysis of PIG and generation of IG and DAG in three CHO cell lines in a dose-dependent manner. Half-maximal concentrations of insulin to induce generation of IG was 8 × 10⁻¹¹ M in the CHO-wt cells, 10⁹ M in the parental CHO cells, and 10⁻⁸ M in the CHO-mut cells. ED₅₀ for the stimulation of DAG generation was 7 × 10⁻¹¹ M in the CHO-wt cells, 10⁻⁹ M in the parental cells, and 10⁻⁸ M in the CHO-mut cells. It is concluded that insulin-dependent PDH activation, PIG hydrolysis, and IG and DAG generation are mediated by the wild-type but not by the mutated insulin receptor of Val⁹⁹⁶. This study suggests that tyrosine kinase activity of the insulin receptor might be a prerequisite for insulin-stimulated generation of IG and DAG.