A forkhead-type transcription factor, DAF-16, is located in the most downstream part of the insulin signalling pathway via PI3K (phosphoinositide 3-kinase). It is essential for the extension of life-span and is also involved in dauer formation induced by food deprivation in Caenorhabditis elegans. In the present study, we addressed whether or not FOXO members AFX, FKHR (forkhead homologue in rhabdomyosarcoma) and FKHRL1 (FKHR-like protein 1), mammalian counterparts of DAF-16, are involved in starvation stress. We found a remarkable selective induction of FKHR and FKHRL1 transcripts in skeletal muscle of mice during starvation. The induction of FKHR gene expression was observed at 6 h after food deprivation, peaked at 12 h, and returned to the basal level by 24 h of refeeding. The induction was also found in skeletal muscle of mice with glucocorticoid treatment. Moreover, we found that the levels of PDK4 (pyruvate dehydrogenase kinase 4) gene expression were up-regulated through the direct binding of FKHR to the promoter region of the gene in C2C12 cells. These results suggest that FKHR has an important role in the regulation of energy metabolism, at least in part, through the up-regulation of PDK4 gene expression in skeletal muscle during starvation.
Abbreviations used: CR, caloric restriction; DBE, DAF-16 family binding element; DMEM, Dulbecco's modified Eagle's medium; FKHR, forkhead homologue in rhabdomyosarcoma; FKHRL1, FKHR-like protein 1; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; G6P, glucose-6-phosphatase; GMSA, gel mobility-shift assay; NEFA, non-esterified fatty acid; PDK4, pyruvate dehydrogenase kinase 4; PEPCK, phosphoenolpyruvate carboxykinase; PI3K, phosphoinositide 3-kinase; PPAR, peroxisome-proliferator-activated receptor; Trx, thioredoxin; TBS, Tris-buffered saline.