Carbohydrate-responsive element-binding protein (ChREBP) is a new transcription factor that binds to the carbohydrate-responsive element of the L-type pyruvate kinase gene (L-PK). The aim of this study was to investigate the mechanism by which feeding high fat diets results in decreased activity of ChREBP in the liver (Yamashita, H., Takenoshita, M., Sakurai, M., Bruick, R. K., Henzel, W. J., Shillinglaw, W., Arnot, D., and Uyeda, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9116 -9121). We cloned the rat liver ChREBP gene for use throughout this study. Acetate, octanoate, and palmitate inhibited the glucose-induced activation of L-PK transcription in ChREBP-overexpressed hepatocytes. In these hepatocytes, the cytosolic AMP concentration increased 30-fold and AMP-activated protein kinase activity was activated 2-fold. Similarly to the fatty acids, 5-amino-4-imidazolecarboxamide ribotide, a specific activator of AMP-activated protein kinase (AMPK) also inhibited the L-PK transcription activity in ChREBPoverexpressed hepatocytes. Using as a substrate a truncated ChREBP consisting of the C-terminal region, we demonstrated that phosphorylation by AMPK resulted in inactivation of the DNA binding activity. AMPK specifically phosphorylated Ser 568 of ChREBP. A S568A mutant of the ChREBP gene showed tight DNA binding and lost its fatty acid sensitivity, whereas a S568D mutant showed weak DNA binding and inhibited L-PK transcription activity even in the absence of fatty acid. These results strongly suggested that the fatty acid inhibition of glucose-induced L-PK transcription resulted from AMPK phosphorylation of ChREBP at Ser 568 , which inactivated the DNA binding activity. AMPK was activated by the increased AMP that was generated by the fatty acid activation.Glucose metabolism in liver is inhibited by administration of fatty acids, the so-called "glucose sparing" effect (1-3). Fatty acids inhibit genes of key enzymes of glycolysis and lipogenesis such as L-pyruvate kinase (L-PK), 1 acetyl-CoA carboxylase, and fatty acid synthetase. L-PK, regulating the flux of metabolites through the pyruvate-phosphoenolpyruvate cycle (4), is known to play an important role in hepatic glucose and lipid metabolism. The activity of L-PK is subject to acute control by covalent modification and allosteric effectors (5). On the other hand, long term control of L-PK is achieved by regulating L-PK gene transcription (5). Fatty acids inhibit transcription of L-PK and other enzymes in glycolysis and lipogenesis pathways, whereas excess glucose induces expression of these genes (6). However, the mechanism of fatty acid inhibition of transcription is not understood. We recently identified a new transcription factor, which binds specifically to the carbohydrate-responsive element of the L-PK gene, and we have termed this new transcription factor, "carbohydrate-responsive element-binding protein" (ChREBP) (7). ChREBP is expressed specifically in liver and is responsive to diet. ChREBP is activated by a high carbohydrate diet and inhibited by a h...
