Signal Transduction Pathways Leading to Insulin-Induced Early Gene Induction

We examined the signal transduction pathway leading to insulin stimulation of two immediate early genes, c-fos, and the early growth response gene, Egr-1. In Rat 1 fibroblasts overexpressing normal human insulin receptors (HIRc-B), insulin and IGF-I rapidly and transiently induced the expression of both c-fos and Egr-1 mRNA with maximum accumulation at 30 min, declining to basal levels at 120 min. Insulin (100 ng/mL) increased c-fos and Egr-1 mRNA expression 10-fold (EC50 = 20 ng/mL), whereas IGF-I (100 ng/mL) and serum (20%) led to a 3- and 11.5-fold increase, respectively. Insulin-stimulated c-fos protein expression was maximal at 1 h postinduction and undetectable at 4 h. The effects of insulin and IGF-I on both c-fos mRNA and protein expression were absent in Rat 1 fibroblasts expressing tyrosine kinase-defective human insulin receptors (A/K1018). In cells expressing insulin receptors in which the two C-terminal tyrosines are mutated to phenylalanine (Y/F2 cells), the insulin stimulated increase in Egr-1 and c-fos mRNA was comparable to that of HIRc cells, whereas, in cells expressing C-terminal truncated receptors (delta CT cells), the insulin induced increase in Egr-1 mRNA was normal, but the c-fos mRNA response was severely blunted. As expected, the insulin effect to increase ras GTP formation and MAP kinase activity was negligible in A/K1018 cells but normal, or supernormal, in Y/F2 cells. Importantly, stimulation of ras GTP was increased in delta CT cells, whereas stimulation of MAP kinase activity was almost absent.(ABSTRACT TRUNCATED AT 250 WORDS)