Expression of the splice variants of the p85 alpha regulatory subunit of phosphoinositide 3-kinase in muscle and adipose tissue of healthy subjects and type 2 diabetic patients

The regulation by insulin of the expression of the p85 alpha regulatory sub unit of phosphoinositide 3-kinase (PI 3-kinase) is impaired in skeletal mus cle and adipose tissue of type 2 diabetic patients. The gene encoding p85a (named grb-1) can generate several variants by alternative splicing, all be ing able to activate the p110 catalytic subunits of PI 3-kinase. Our aims w ere (i) to determine the mRNA expression profiles of these variants in huma n skeletal muscle and adipose tissue (ii) to investigate the effect of insu lin on their expression in vivo and in vitro in muscle and (iii) to verify whether this regulation is defective in type 2 diabetes. We determined the human genomic organization of grb-1 and set up reverse transcriptase compet itive PCR assays for the quantification of each mRNA variant. In muscle, p8 5 alpha and p50 alpha mRNAs were the most abundant, and p55 alpha represent ed less than 20% of all grb-1-derived mRNAs. In adipose tissue, p85 alpha w as expressed predominantly and p55 alpha mRNA was not detectable. These exp ression profiles were not different in type 2 diabetics. During a 3 h hyper insulinaemic clamp, insulin increased the mRNA expression of the three vari ants in muscle of control subjects. In diabetic patients, the effect of ins ulin on p85 alpha and p50 alpha mRNAs was blunted, and largely reduced on p 55 alpha transcripts. In cultured human myotubes, up-regulation of p85 alph a, p55 alpha and p50 alpha mRNAs by insulin was abolished by LY294002 (10 m uM) and by rapamycin (50 nM), suggesting that the PI 3-kinase/protein kinas e B/p70 S6 kinase pathway could be involved in the stimulation of grb-1 gen e expression by insulin in human muscle cells.