Natural variants of human p85 alpha 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 prev iously reported. Methods. Phosphoinositide 3-kinase p85 alpha regulatory subunit cDNA was ex amined in 20 subjects with syndromes of severe insulin resistance by single strand conformational polymorphism and restriction fragment length polymor phism analyses. Insulin-stimulated phosphoinositide 3-kinase activity and r ecruitment into phosphotyrosine complexes of variants of p85 alpha were stu died in transiently transfected HEK293 cells. Phosphopeptide binding charac teristics of wild-type and mutant p85 alpha-GST fusion proteins were examin ed 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 phosphotyrosi ne recruitment as wild-type p85 alpha. A novel heterozygous mutation, Arg(4 09)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) com pared with wild-type relatives (72 mol/l) (n = 8 subjects, p = 0.06). The A rg409Gln p85 alpha subunit was associated with lower insulin-stimulated pho sphoinositide 3-kinase activity compared with wild-type (mean reduction 15% , p < 0.05, n = 5). The recruitment of Arg409Gln p85 alpha into phosphotyro sine complexes was not significantly impaired. GST fusion proteins of wild- type and mutant p85 alpha showed identical binding to phosphopeptides in su rface 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 vari ant appears to have no functional effect on the insulin-stimulated phosphoi nositide 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.