Induction of gluconeogenesis is a vital adaptation to fasting. However, excessive hepatic glucose production (HGP) also contributes to the pathophysiology of diabetes. In healthy individuals, post-prandial glycemic excursions are limited by release of insulin from pancreatic islets. Insulin lowers blood glucose levels by directly stimulating glucose uptake and also by inhibiting HGP. The Forkhead Box-containing, sub-family O protein 1 (FoxO1) is the primary mediator of the transcriptional regulation of gluconeogenesis. During fasting conditions FoxO1 is found in the nucleus, while in response to insulin receptor signaling FoxO1 is phosphorylated and rapidly translocates to the cytoplasm for inactivation by proteasomal degradation. Evidence from our lab and others has shown that the transcriptional activity of FoxO1 is further modified by protein acetylases and deacetylases such as CBP/p300 and SirT1, respectively. We hypothesize that metabolic stress accrued though ageing and obesity can activate FoxO1 through deacetylation thereby contributing to the pathogenesis of diabetes. The silent information regulator 2 (Sir2 alpha) is a NAD+ dependent protein deacetylase that controls longevity in fruit flies and nematodes in a FoxO1- dependent manner. Sirtl, Sir2's mammalian ortholog, regulates FoxO1 and PGC-1 alpha in hepatocytes, thus leading to the suggestion that it modulates glucose production. We therefore propose to examine the effects of Sirtl on metabolism in mice.
In Aim 1, we will utilize a Sirtl BAG to generate transgenic mice (SirT1-Tg) with low levels of over-expression, similar to those that have been shown to affect life span in C. elegans. We hypothesize that Foxol is required to mediate the salutary effects of SirT1. To test this theory, we will examine whether the metabolic phenotype of SirT1-Tg mice is caused by its effects on FoxO1 or PGC-1 alpha deacetylation.
In Aim 2, we propose to replace the wild-type FoxO1 gene with alleles encoding either acetylation-defective or constitutively acetylated mutants to asses the effects on insulin signaling and gluconeogenesis. Relevance: The mechanism of insulin action on gene expression is a key question in biology with important ramifications for the treatment of metabolic disorders. In lower eukaryotes, strong evidence exists to support the relationship between the insulin/IGF1 signaling pathway and regulation of metabolism and lifespan. By genetically amplifying the SirT1 gene, and rendering its downstream target, FoxO1 constitutively active we hope to improve glycemic control and possibly lifespan in mice. ? ? ?
