Natural variants of human p85α phosphoinositide 3-kinase in severe insulin resistance:: a novel variant with impaired insulin-stimulated lipid kinase activity

Aims/hypothesis. Phosphoinositide 3-kinase (PI 3K) plays a central part in the mediation of insulin-stimulated glucose disposal. No genetic studies of this enzyme in human syndromes of severe insulin resistance have been previously reported. Methods. Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was examined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymorphism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and recruitment into phosphotyrosine complexes of variants of p85 alpha were studied in transiently transfected HEK293 cells. Phosphopeptide binding characteristics of wild-type and mutant p85 alpha-GST fusion proteins were examined by surface plasmon resonance. Results. The common p85 alpha variant, Met(326)Ile, was identified in 9 of the 20 subjects. Functional studies of the Met(326)Ile variant showed it to have equivalent insulin-stimulated lipid kinase activity and phosphotyrosine recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg(409)Gln, was detected in one subject. Within the proband's family, carriers of the mutation had a higher median fasting plasma insulin (218 pmol/l) compared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The Arg409Gln p85 alpha subunit was associated with lower insulin-stimulated phosphoinositide 3-kinase activity compared with wild-type (mean reduction 15%, p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyrosine complexes was not significantly impaired. GST fusion proteins of wild-type and mutant p85 alpha showed identical binding to phosphopeptides in surface plasmon resonance studies. Conclusion/interpretation. Mutations in p85 alpha are uncommon in subjects with syndromes of severe insulin resistance. The Met(326)Ile p85 alpha variant appears to have no functional effect on the insulin-stimulated phosphoinositide 3-kinase activity. The impaired phosphoinositide 3-kinase activity of the Arg(409)Gln mutant suggests that it could contribute to the insulin resistance seen in this family.