Genetic evidence in worms (C. Elegans) has shown that insulin-like molecules act via PI3 kinase and AKT/protein kinase B to inhibit the function of the forkhead (FKH) transcription factor DAF-16. Insulin signaling mutants with diminished function undergo dauer arrest and show increased longevity and resistance to oxidative stress due to the unimpeded action of DAF-16. Accordingly, DAF-16 has been shown to activate the superoxide dismutase gene (SOD). In mammals, caloric restriction and low insulin signaling has been shown to slow the rate of aging by mechanisms that include increased DNA repair capacity and reduction of oxidative stress. In the presence of low glucose and circulating insulin levels, DAF-16 homologues appear to be transcriptionally active. Several laboratories have shown that in the absence of insulin, mammalian homologues of DAF-16, FKHR, FKHRL1 and AFX activate the transcription of genes that control apoptosis, and gluconeogenesis, and insulin can inhibit this effect. Our goal is to elucidate the mechanisms by which DAF-16 like factors activate transcription in the absence of insulin. Caloric restriction and low glucose activates AMP kinase. We find that AMP kinase can prevent the effect of insulin on DAF-16 in HepG2 cells. Furthermore, AMP kinase can directly phosphorylate DAF-16. We propose to determine whether regulation of DAF-16 by AMP kinase in worms and regulation of its homologue FKHR in mammalian cells, can explain the ability of caloric restriction to slow the aging process. In HepG2 cells, insulin signaling via the AKT sites in DAF-16 inhibits DAF-16 activity. We find that the AKT sites in DAF-16 carry overlapping AMP kinase sites.
In Specific Aim 1 of this proposal, we will determine whether AMP kinase regulates DAF-16 activity directly by altering its phosphorylation or indirectly by regulating other elements of the PI3 kinase-signaling pathway. We will examine the effect of AMP kinase on 1) phosph DAF-16 phosphorylation in vitro and in vivo, 2) 14-3-3 binding to DAF-16 in the presence and absence of insulin and 3) the interaction of DAF-16 with other proteins that increase its binding/transcription activity.
In Specific Aim II we will determine whether AMP kinase can counteract the effect of insulin signaling to DAF-16 in C. elegans, and prolong life span in the worm.
In Specific Aim III, we will examine the effect of caloric restriction and carbohydrate-induced hyperinsulinemia on the activity of AMP kinase and DAF-16 homologues
