PI 3-kinase activation is central to most of the metabolic actions of insulin (3
). The major regulatory subunits of PI 3-kinase in most cells are p85α and its alternatively spliced isoforms AS53/p55α and p50α encoded by the Pik3r1
gene. In the present study, we have shown that reducing the level of p85α, AS53, and p50α by heterozygous disruption of the Pik3r1
gene results in increased insulin sensitivity, lower fasting and postprandial glucose levels, and a significant decrease in the incidence of diabetes related to genetically induced insulin resistance in mice.
Recently, Terauchi et al. reported that selective homozygous disruption of the p85α full-length form of regulatory subunit results in hypoglycemia and increased sensitivity to insulin (20
). The authors suggested that this was a result of an isoform switch from p85α to p50α or AS53 in insulin-sensitive tissues and that p50α is more potent in activation of PI 3-kinase–dependent insulin signaling than is p85α. Since the expression levels of p85α, p50α, and AS53 are all reduced in the Pik3r1
knockout mouse, this mechanism cannot be responsible for the increased insulin sensitivity observed in the present study. Rather, changes in the molecular balance between p85 and p110 by heterozygous knockout of Pik3r1
appear to play a key role in this phenomenon. Indeed, immunodepletion studies reveal that in liver of wild-type mouse, 30% of p85 exits as a monomer that may compete with p85-p110 dimer in binding to phosphorylated IRS proteins, thereby inhibiting PI 3-kinase–dependent signaling. Heterozygous disruption of Pik3r1
decreases p85 monomer with only slight reduction of the amount of p85-p110 dimer. As a consequence, p85+/–
mice group exhibits PI 3-kinase activities almost equal to those of mice with normal levels of p85α. Thus, heterozygous disruption of Pik3r1
increases the ratio of p85-p110 dimer to p85 monomer. This could improve insulin-induced PI 3-kinase signaling, depending on the balance between p85 and phosphorylated IRS proteins. For instance, if phosphorylated IRS proteins are less abundant than total p85 monomer and p85-p110 dimer in a p85+/–
tissue, the increase in the ratio of p85-p110 dimer to p85 monomer by heterozygous disruption of Pik3r1
could improve PI 3-kinase signaling by increasing the amount of p85/p110/IRS complexes. However, if phosphorylated IRS proteins are more abundant than total p85 in wild-type, the increase of the ratio by heterozygous disruption of Pik3r1
might not affect PI 3-kinase–dependent signaling, because all of the p85-p110 dimers can bind IRS proteins even in the wild-type.
Following insulin injection, the improvement of PI 3-kinase–dependent signaling in mice with a heterozygous disruption of Pik3r1 was confirmed by increased Akt activity, at least in wild-type and IR/IRS-1+/– mice. Since under normal feeding conditions, phosphorylation levels of IRS proteins should be much lower than those caused by pharmacological insulin injection, the improvement of PI 3-kinase–dependent signaling leading to improved insulin sensitivity would be even more pronounced.
Heterozygous disruption of Pik3r1
also results in an increase in the PI 3-kinase activity associated with p85β, at least in liver of the mouse, and this may also contribute to the increased insulin sensitivity. We (24
) and others (23
) have shown that p85α inhibits the lipid kinase activity of the p110 catalytic subunit in vitro. p85β appears to be less potent in this negative regulation, as evidenced by the finding that in Pik3r1
-null mice PI 3-kinase activity associated with p85β maintains Akt activation, even though p85α is completely absent and p110 is markedly decreased (22
In addition, p85α may modulate PI 3-kinase–dependent signaling by effects that are independent of its regulation of p110. For example, we find that in cultured cells derived from p85+/–
mice, PI(3, 4, 5)P3
production is increased and the levels more sustained as compared with those in the wild-type cells, even though PI 3-kinase activity associated with phosphotyrosine complexes is equal in these two cell types (36
). This suggests that p85α protein may have some effect to modulate PI(3, 4, 5)P3
clearance independent of its effect on PI 3-kinase activity (36
). This could account for the 30% increase in Akt activity in livers of p85+/–
mice, since Akt activity depends on PI(3, 4, 5)P3
levels. Similarly, Terauchi et al. demonstrated that disrupting the full-length p85α alone, results in an increased and sustained PI(3, 4, 5)P3
production, although PI 3-kinase activity associated with phosphotyrosine complex is markedly decreased (20
While the effect of reducing p85α levels on insulin sensitivity is clear, it remains to be determined which tissue is most important in this physiological response. In both isolated skeletal muscle (this study) and cultured brown adipocyte cell lines (K. Ueki et al., unpublished data), basal glucose uptake is increased in the p85+/–
tissues as compared with controls, although no significant difference is observed in insulin-stimulated glucose uptake. This could contribute to lower fasting glucose levels. Lower fasting glucose levels may also reflect increased sensitivity to insulin-induced suppression of hepatic glucose output, since the enzyme phosphoenolpyruvate carboxykinase (PEPCK) is negatively regulated by insulin via a PI 3-kinase/Akt–mediated pathway (14
), and increasing Akt activity in liver has been shown to lower glucose levels in insulin-resistant mice (28
). Thus, insulin-induced inhibition of hepatic glucose production seems to be important for whole-body insulin sensitivity observed in p85+/–
mice. However, in preliminary studies, we found no change in mRNA levels of PEPCK in the p85α heterozygous mice (data not shown). It is possible that any differences in PEPCK are too small to be detected or that the increased Akt activity in liver modifies other enzymes involved in hepatic glucose production, such as 6-phospho-fructose 2-kinase (38
) and glycogen synthase kinase 3 (39
). Further studies using cultured cells heterozygous for the Pik3r1
-null allele would help characterize these issues.
In summary, reducing the expression level of p85α by 50% significantly improves insulin sensitivity and results in a decrease in the incidence of diabetes in mouse models of insulin resistance. This appears to be due to an improved stoichiometry of the p85/p110/IRS complex and enhanced PI 3-kinase–dependent signaling. Thus p85α can play a dual role in insulin action mediating PI 3-kinase activation by bridging IRS proteins and the catalytic p110 subunit, but it can also act as a competitive inhibitor in PI 3-kinase signaling in its monomeric state. Pharmacological modulation of p85α expression in insulin-sensitive tissues appears to be a novel strategy to improve insulin sensitivity and may serve as a new therapeutic target in the treatment of type 2 diabetes.