👤 T Kabashima

Carbohydrate-responsive element binding protein (ChREBP) is a transcription factor that activates lipogenic genes in liver in response to excess carbohydrate in the diet. ChREBP is regulated in a reciprocal manner by glucose and cAMP. cAMP-dependent protein kinase (protein kinase A) phosphorylates two physiologically important sites in ChREBP, Ser-196, which is located near nuclear localization signal sequence (NLS), and Thr-666, within the basic helix-loop-helix (bHLH) site, resulting in inactivation of nuclear translocation of ChREBP and of the DNA-binding activity, respectively. We demonstrate here that crude cytosolic extracts from livers of rats fed a high carbohydrate diet contained protein phosphatase (PPase) activity that dephosphorylated a peptide containing Ser-196, whereas a PPase in the nuclear extract catalyzed dephosphorylation of Ser-568 and Thr-666. All these PPases are activated specifically by xylulose 5-phosphate (Xu5P), but not by other sugar phosphates. Furthermore, addition of Xu5P elevated PPase activity to the level observed in extracts of fed liver cells. These partially purified PPases were characterized as PP2A-AB delta C by immunoblotting with specific antibodies. These results suggest that (ia) Xu5P-dependent PPase is responsible for activation of transcription of the L-type pyruvate kinase gene and lipogenic enzyme genes, and (ii) Xu5P is the glucose signaling compound. Thus, we propose that the same Xu5P-activated PPase controls both acute and long-term regulation of glucose metabolism and fat synthesis. Show less

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 ChREBP-overexpressed 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. Show less

Recently we purified and identified a previously uncharacterized transcription factor from rat liver binding to the carbohydrate responsive element of the L-type pyruvate kinase (L-PK) gene. This factor was named carbohydrate responsive element binding protein (ChREBP). ChREBP, essential for L-PK gene transcription, is activated by high glucose and inhibited by cAMP. Here, we demonstrated that (i) nuclear localization signal and basic helix-loop-helix/leucine-zipper domains of ChREBP were essential for the transcription, and (ii) these domains were the targets of regulation by cAMP and glucose. Among three cAMP-dependent protein kinase phosphorylation sites, Ser(196) and Thr(666) were the target sites. Phosphorylation of the former resulted in inactivation of nuclear import, and that of the latter resulted in loss of the DNA-binding activity and L-PK transcription. On the other hand, glucose activated the nuclear import by dephosphorylation of Ser(196) in the cytoplasm and also stimulated the DNA-binding activity by dephosphorylation of Thr(666) in the nucleus. These results thus reveal mechanisms for regulation of ChREBP and the L-PK transcription by excess carbohydrate and cAMP. Show less