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. 2008 Aug 29;283(35):24029-38.
doi: 10.1074/jbc.M801539200. Epub 2008 Jun 30.

Glucose activates ChREBP by increasing its rate of nuclear entry and relieving repression of its transcriptional activity

Michael N Davies  1 , Brennon L O'Callaghan, Howard C Towle
Affiliations

Glucose activates ChREBP by increasing its rate of nuclear entry and relieving repression of its transcriptional activity

Michael N Davies et al. J Biol Chem. .

Abstract

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that activates genes involved in de novo lipogenesis in mammals. The current model for glucose activation of ChREBP proposes that increased glucose metabolism triggers a cytoplasmic to nuclear translocation of ChREBP that is critical for activation. However, we find that ChREBP actively shuttles between the cytoplasm and nucleus in both low and high glucose in the glucose-sensitive beta cell-derived line, 832/13. Glucose stimulates a 3-fold increase in the rate of ChREBP nuclear entry, but trapping ChREBP in the nucleus by mutagenesis or with a nuclear export inhibitor does not lead to constitutive activation. In fact, mutational studies targeting the nuclear export signal of ChREBP also identified a distinct function essential for glucose-dependent transcriptional activation. From this, we conclude that an additional event independent of nuclear translocation is required for activation. The N-terminal segment of ChREBP (amino acids 1-298) has previously been shown to repress activity under basal conditions. This segment has five highly conserved regions, Mondo conserved regions 1-5 (MCR1 to -5). Based on activating mutations in MCR2 and MCR5, we propose that these two regions act coordinately to repress ChREBP in low glucose. In addition, other mutations in MCR2 and mutations in MCR3 were found to prevent glucose activation. Hence, we conclude that both relief of repression and adoption of an activating form are required for ChREBP activation.

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Figures

FIGURE 1.
FIGURE 1.
ChREBP shuttles between the cytoplasm and nucleus under both low and high glucose conditions. a, 832/13 cells were co-transfected with expression plasmids for FLAG-tagged ChREBP and Mlx overnight in 11 mm glucose and subsequently incubated in RPMI medium containing 2.5 mm (low) glucose for 4 h. Cells were then continued in the same medium or switched to RPMI medium containing 25 mm (high) glucose for 2 h. Immunofluorescence was performed using an anti-FLAG primary antibody and a FITC-conjugated secondary antibody, and images were obtained by confocal microscopy. FITC immunofluorescence represents ChREBP localization and is shown in green. Nuclei were stained with TO-PRO3 and are shown in blue. The panels labeled FITC represent ChREBP localization, whereas the panels labeled FITC + Nuclei show both ChREBP localization and nuclear staining. b, cells were treated as described above, except that leptomycin B was added simultaneously with the glucose treatment for 2 h.
FIGURE 2.
FIGURE 2.
The rate of ChREBP nuclear entry is increased in high glucose conditions. a, 832/13 cells were co-transfected with FLAG-tagged ChREBP and Mlx overnight in RPMI medium containing 11 mm glucose and were then incubated in low glucose medium for 4 h. Cells were then treated with either low or high glucose for 2 h, and immunofluorescence was performed as described under “Experimental Procedures.” Over 100 cells in each treatment were scored as predominantly cytoplasmic, both cytoplasmic and nuclear, or predominantly nuclear by two observers. Results represent the means ± S.E. for three experiments. b, cells were treated with low (open triangles) or high glucose (closed squares) medium with the addition of leptomycin B at various time points as indicated. Immunofluorescence and quantification was performed as described above. Cells displaying either predominantly nuclear or both nuclear and cytoplasmic localization were combined and expressed as a percentage of the total cells.
FIGURE 3.
FIGURE 3.
The L86A/L93A mutant of ChREBP is trapped in the nucleus but is transcriptionally inactive. a, immunofluorescence images of 832/13 cells co-transfected with FLAG-tagged L86A/L93A ChREBP mutant and Mlx in low and high glucose conditions are shown. See the legend to Fig. 1 for details. b, a functional assay for ChREBP activity was performed in 832/13 cells. 832/13 cells were transduced with an adenoviral vector expressing dominant negative ChREBP. Cells were then co-transfected with a luciferase reporter plasmid containing two copies of the ACC ChoRE, a Renilla luciferase reporter, and expression plasmids for either WT ChREBP or the L86A/L93A ChREBP mutant and WT Mlx. After 18 h, cells were treated with low or high glucose for 24 h, and extracts were prepared. Values are in relative light units (firefly/Renilla) and represent the means ± S.D. for triplicate samples.
FIGURE 4.
FIGURE 4.
Leptomycin B does not inhibit the induction of ChREBP target genes in 832/13 cells. 832/13 cells were pretreated in low glucose RPMI medium for 4 h with or without leptomycin B (Lep) for the last 90 min. Cells from both experimental groups were then incubated with low or high glucose for 4 h. RNA was isolated and converted into cDNA using reverse transcriptase. mRNA levels of ChREBP target genes, thioredoxin-binding protein (Txnip) and aldolase B (AldoB), were measured by quantitative reverse transcription-PCR. mRNA levels in low glucose without the addition of leptomycin B were set to 1, and all values are normalized to this group. Values represent the mean ± S.E. of triplicate samples.
FIGURE 5.
FIGURE 5.
NES region mutants of ChREBP separate NES function from transcriptional activation. a, immunofluorescence images of 832/13 cells transfected with FLAG-tagged L89A, F90A, and L95A ChREBP mutants and Mlx in low and high glucose conditions are shown. See the legend to Fig. 1 for details. b, functional activities of ChREBP mutants were tested using the reporter assay as described in the legend to Fig. 3b. Values are shown as relative light units (firefly/Renilla) and represent the means ± S.D. for triplicate samples.
FIGURE 6.
FIGURE 6.
MCR5 and MCR2/MCR5 mutants of ChREBP display increased transcriptional activity. a, conserved regions in the amino-terminal segment of ChREBP. The locations of five highly conserved regions in the amino-terminal segment of ChREBP are indicated. Numbering is based on mouse ChREBP. These regions are highly conserved (>90% identity) with the ChREBP paralog, MondoA, as well as with ChREBP orthologs from pufferfish to human. Previously proposed functions for these conserved domains are indicated below. GSD, glucose-sensing domain; NLS, nuclear localization signal; LID, low glucose inhibitory domain; GRACE, glucose response activation conserved element. b, functional activity of ChREBP was tested using the rescue assay described in the legend to Fig. 3b. WT, F90A, Y275A/V276A/G277A (275-277), L289A/Q290A/P291A (289-291), F90A/275-277, and F90A/289-291 were introduced and tested for activity. Values are shown as relative light units (firefly/Renilla) and represent means ± S.D. for triplicate samples. c, luciferase values measured in low glucose conditions of the mutants above are compared. The data presented in this panel is from the experiment shown in b.*, p < 0.05 compared with WT ChREBP; **, p < 0.01 compared with WT ChREBP.
FIGURE 7.
FIGURE 7.
MCR3 mutants of ChREBP inhibit glucose activation. a, functional activity of ChREBP was tested using the reporter assay described in the legend to Fig. 3b. WT ChREBP or ChREBP mutants N123F, I126Q, and W130A were introduced and tested for activity. Values are shown as relative light units (firefly/Renilla) and represent the means ± S.D. for triplicate samples. b, 832/13 cells were co-transfected with FLAG-tagged WT or mutant ChREBP and Mlx and incubated overnight in RPMI medium with 11 mm glucose. Cell extracts were prepared, and co-immunoprecipitations were performed with anti-FLAG immunobeads as described under “Experimental Procedures.” Immunoblotting was carried out with a 14-3-3β antibody. Lane 1, extracts from untransfected 832/13 cells. Lanes 2-5, cells transfected with WT ChREBP and MCR3 mutations N123F, I126Q, and W130A, respectively. Lane 6, an aliquot of the cell extract without co-immunoprecipitation. The arrow indicates the position of 14-3-3β protein, and the asterisk indicates a background band that cross-reacts with the 14-3-3β antibody.
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