Class IA phosphoinositide (PI) 3-kinase is composed of a p110 catalytic subunit and a p85 regulatory subunit and plays a pivotal role in insulin signaling. To explore the physiological roles of two major regulatory isoforms, p85␣ and p85␤, we have established brown adipose cell lines with disruption of the Pik3r1 or Pik3r2 gene. Pik3r1 ؊/؊ (p85␣ ؊/؊ ) cells show a 70% reduction of p85 protein and a parallel reduction of p110. These cells have a 50% decrease in PI 3-kinase activity and a 30%

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... ease in Akt activity, leading to decreased insulininduced glucose uptake and anti-apoptosis. Pik3r2 ؊/؊ (p85␤ ؊/؊ ) cells show a 25% reduction of p85 protein but normal levels of p85-p110 and PI 3-kinase activity, supporting the fact that p85 is more abundant than p110 in wild type. p85␤ ؊/؊ cells, however, exhibit significantly increased insulin-induced Akt activation, leading to increased anti-apoptosis. Reconstitution experiments suggest that the discrepancy between PI 3-kinase activity and Akt activity is at least in part due to the p85-dependent negative regulation of downstream signaling of PI 3-kinase. Indeed, both p85␣ ؊/؊ cells and p85␤ ؊/؊ cells exhibit significantly increased insulin-induced glycogen synthase activation. p85␣ ؊/؊ cells show decreased insulin-stimulated Jun N-terminal kinase activity, which is restored by expression of p85␣, p85␤, or a p85 mutant that does not bind to p110,