Xylulose 5-phosphate mediates glucose-induced lipogenesis by xylulose 5-phosphate-activated protein phosphatase in rat liver

Abstract

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.

Figure 1

The domain structure of ChREBP. The locations of the NLS, proline-rich stretch, bHLH/leucine zipper (bHLH/ZIP), and ZIP-like domains are indicated. The locations of three phosphorylation sites of PKA are designated as P1, P2, and P3. The AMP-activated protein kinase (AMPK) site is indicated as P4.

Figure 2

Dephosphorylation of P1 site peptide by PPase in cytosol. (A) PPase activity in the cytosolic extracts of livers from fasted rats vs. high-carbohydrate-fed rats. The PPase activity was determined with the assay method described in Materials and Methods with the P1 site peptide as a substrate. Crude cytosolic extract (5 μg) was added to the reaction mixture, incubated for 15 min at 30°C, and assayed for [³²P]phosphate released. The PPase activity was expressed relative to that in the fasted liver extract, which was 100%. (B) Activation of desalted crude extracts by various metabolites. To remove endogenous metabolites the crude extracts were filtered through Sephadex G50 by centrifugation, and the filtered extract (5 μg) was assayed for the PPase activity in the presence of 50 μM sugar phosphates. G6P, glucose 6-phosphate; F6P, fructose 6-phosphate; 3PG, 3-phosphoglycerate; PEP, phosphoenolpyruvate; Ru5P, ribulose 5-phosphate; R5P; ribose 5-phosphate; 6PG, 6-phosphogluconate.

Figure 3

(A) DEAE-cellulose chromatography of Xu5P-activated PPase in cytosol. The cytosolic extract (1,500 mg) from rat liver was chromatographed on a DEAE-cellulose column (3 cm × 10 cm) as described previously (15). The PPase activity was assayed with P1 peptide as a substrate in the absence (○) or presence (●) of 50 μM Xu5P. □, A₂₈₀. (B) Mono Q chromatography of the Xu5P-activated PPase eluted from the DEAE-cellulose column in A. The fractions (48–56) were pooled and loaded on a Mono Q column (0.5 cm × 5 cm). PPase was eluted with a linear gradient of 0–0.5 M NaCl in 50 mM Tris⋅HCl/0.1 mM EDTA/1 mM DTT. (C) Immunoblot of Xu5P-activated PPase eluted from the Mono Q column. The fractions (22–25, 15 μl each) were subjected to PAGE, electroblotted onto a nitrocellulose filter, and incubated with primary antibodies against PP2A A, Bα, Bδ, and C.

Figure 4

PPase activity in nuclear extract. P3 peptide was used as substrate. (A) PPase activity in crude nuclear extracts (5 μg) from fasted and high-carbohydrate-fed rat livers was assayed as in Fig. 2. (B) Various sugar phosphates (50 μM) were added to the assay mixture and PPase activity was determined as in Fig. 2B. The same results were obtained with P4 peptide as a substrate (data not shown).

Figure 5

(A and B) DEAE-cellulose and Mono Q chromatography of Xu5P-activated PPase in nuclear extract. Crude nuclear extract (800 mg) was subjected to DEAE-cellulose (A) followed by Mono Q (B) chromatography as in Fig. 3A. The PPase activity was assayed with P3 as a substrate in the absence (○) or presence (●) of 50 μM Xu5P. (C) Immunoblots of PPase eluted from the Mono Q column as in Fig. 3C. Fractions 22–25 (15 μl) were subjected to PAGE, electroblotting, and immunoreaction as above.

Figure 6

Kinetics of Xu5P formation, nuclear import, and the LPK transcription activity in primary hepatocytes. The hepatocytes transfected with either GFP-ChREBP or ChREBP were incubated in low (open symbols, 5.5 mM) and high (filled symbols, 27.5 mM) concentrations of glucose. At indicated time intervals, the hepatocytes were harvested. An aliquot was immediately treated with HClO₄ for metabolite determination, and the remainder was used for assay of translocation and LPK transcription, as described in Materials and Methods. ●, Xu5P formation; ▴, nuclear import; ■, LPK transcription.

Figure 7

Abbreviated glycolysis, gluconeogenesis, and pentose shunt pathways and roles of Xu5P and Fru-2,6-P₂ in activation of PP2A and PFK, respectively. The scheme illustrates the formation of Xu5P from fructose 6-phosphate (F6P) and glyceraldehyde 3-phosphate (GAP) by transketolase, which activates PP2A. Xu5P PP2A then activates 6-phosphofructo-2-kinase/Fru-2,6-P₂ phosphatase (PF2K/Pase) to increase Fru-2,6-P₂, resulting in activation of PFK. The same PP2A also activates ChREBP in the activation of lipogenic gene transcription, including ATP citrate lyase (ACL), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS). Glu, glucose; HMP, hexose monophosphate; TCA, tricarboxylic acid cycle.