The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity

Abstract

The forkhead transcription factor FoxO1 has been identified as a negative regulator of insulin/IGF-1 signaling. Its function is inhibited by phosphorylation and nuclear exclusion through a PI3K-dependent pathway. However, the structure/function relationship of FoxO1 has not been elucidated completely. In this study, we carried out mutation analysis of the FoxO1 coactivator-interacting LXXLL motif (amino acids 459-463). Expression of a 3A/LXXAA mutant, in which 3 Akt phosphorylation sites (T24, S253, and S316) and 2 leucine residues in the LXXLL motif (L462 and L463) were replaced by alanine, decreased both Igfbp-1 and G6Pase promoter activity and endogenous Igfbp-1 and G6Pase gene expression in simian virus 40-transformed (SV40-transformed) hepatocytes. Importantly, mutagenesis of the LXXLL motif eliminated FoxO1 interaction with the nicotinamide adenine dinucleotide-dependent (NAD-dependent) deacetylase sirtuin 1 (Sirt1), sustained the acetylated state of FoxO1, and made FoxO1 nicotinamide and resveratrol insensitive, supporting a role for this motif in Sirt1 binding. Furthermore, intravenous administration of adenovirus encoding 3A/LXXAA FoxO1 into Lepr db/db mice decreased fasting blood glucose levels and improved glucose tolerance and was accompanied by reduced G6Pase and Igfbp-1 gene expression and increased hepatic glycogen content. In conclusion, the LXXLL motif of FoxO1 may have an important role for its transcriptional activity and Sirt1 binding and should be a target site for regulation of gene expression of FoxO1 target genes and glucose metabolism in vivo.

Increased Expression and Activity of the Transcription Factor FOXO1 in Nonalcoholic Steatohepatitis

“…To check this hypothesis, we measured the expression of FOXO1 and interacting nuclear factors (20). We observed a progressive increase in FOXO1 mRNA levels from subjects with normal liver to those with steatosis alone and NASH, and a significant correlation between FOXO1 mRNA and protein levels.…”

“…Furthermore, downregulation of FOXO1 by antisense oligonucleotides reduced hepatic insulin resistance in a model of diet-induced obesity and fatty liver (18). Although NAFLD is characterized by hyperinsulinemia, under oxidative stress conditions, as in those observed in NASH (4), FOXO1 becomes unresponsive to insulin because of interaction with the deacetylase sirtuin 1, resulting in induction of glucogenetic genes (19,20). Therefore, deregulation of hepatic FOXO1 and the associated transcriptional machinery in response to steatosis and oxidative stress may play a role in the metabolic derangement of subjects with NAFLD by inducing hepatic glucose output.…”

Pck1 Gene Silencing in the Liver Improves Glycemia Control, Insulin Sensitivity, and Dyslipidemia in db/db Mice

“…A nutrient signaling response mediated by pyruvate induces Sirt1 protein in liver during fasting, where it interacts with and deacetylates PGC-1␣ in an NAD ϩ -dependent manner, increasing PGC-1␣ ability to coactivate HNF-4␣ and, therefore, upregulate gluconeogenic genes, but not mitochondrial genes (20). In addition, Sirt1-mediated deacetylation of FOXO1 has been shown to promote its transcriptional activity over gluconeogenic genes (39,40), suggesting an integrated regulation by insulin and Sirt1 over gluconeogenesis in treated animals. Moreover, Sirt1 transcription is inhibited by a NADH-mediated mechanism (41).…”

The FoxO transcription factors and metabolic regulation

“…The LXXLL motif of FoxO1 appears to be important for its binding to Sirt1. Although it has not been elucidated completely how the LXXLL motif of FoxO1 mediates the binding to Sirt, the disruption of the LXXLL motif of FoxO1 inhibits transcriptional activity of FoxO1 and enhance acetylation of FoxO1 in liver [51].…”