Nuclear accumulation of β-catenin, a widely recognized marker of poor cancer prognosis, drives cancer cell proliferation and senescence bypass and regulates incretins, critical regulators of fat and glucose metabolism. Diabetes, characterized by elevated blood glucose levels, is associated with increased cancer risk, partly because of increased insulin growth factor 1 signaling, but whether elevated glucose directly impacts cancer-associated signal-transduction pathways is unknown. Here, we show that high glucose is essential for nuclear localization of β-catenin in response to Wnt signaling. Glucose-dependent β-catenin nuclear retention requires lysine 354 and is mediated by alteration of the balance between p300 and sirtuins that trigger β-catenin acetylation. Consequently β-catenin accumulates in the nucleus and activates target promoters under combined glucose and Wnt stimulation, but not with either stimulus alone. Our results reveal a mechanism by which high glucose enhances signaling through the cancer-associated Wnt/β-catenin pathway and may explain the increased frequency of cancer associated with obesity and diabetes.

Here, we show that nuclear accumulation of stabilized β-catenin requires glucose in a wide range of human tumor-derived cell lines. Upon Wnt stimulation, glucose promotes the formation of a LEF-1/β-catenin complex that associates with the acetylase p300 and displaces the SIRT1 deacetylase, leading to increased β-catenin acetylation, its nuclear accumulation, and transcription activation. Consistently, the lysine 354 mutation in β-catenin abolishes glucose amplification of Wnt-dependent transcription. These results highlight a key mechanism that ties glucose levels to Wnt/β-catenin signaling, with important implications for both cancer and glucose homeostasis.

Wnt-induced inactivation of Glycogen synthase kinase 3β (GSK3β) leads to β-catenin stabilization () and is required for β-catenin entry into the nucleus, where it binds members of the T cell factor (TCF)/lymphoid enhancer factor (LEF) such as LEF-1 to activate the transcription of target genes (). The mechanisms that allow nuclear accumulation of β-catenin are poorly understood.

The connections between Wnt signaling and diabetes, although increasingly recognized, are not straightforward. Upon glucose ingestion, the body responds by secreting insulin to lower blood glucose levels; failure to do so leads to diabetes. The earliest response to glucose ingestion is secretion by enteroendocrine cells of incretin hormones, which are required for normal glucose-dependent insulin secretion and also act on extrapancreatic tissues to control the global body energy balance (). Glucose-dependent insulinotropic peptide (GIP) accounts for 75% of incretin plasma levels () and is responsible for pancreatic glucose-dependent insulin secretion; its signaling is lost in diabetes (). Wnt/β-catenin increases the expression of incretin genes GIP and GCG (proglucagon) in enteroendocrine cells, reviewed in, and modulates incretin signaling in pancreatic beta cells (). Thus, control of global metabolism by Wnt signaling might be mediated at least partially through increased expression and signaling of incretins.

The Wnt/β-catenin pathway is a global regulator of embryonic development, is required for tissue renewal in postembryonic animals (for example, in the maintenance of stem cells in intestinal crypts []), and, when deregulated, can promote senescence bypass (), aberrant cell growth, and cancer (). As such, nuclear accumulation of β-catenin is one of the most widely recognized markers of malignancy. Significantly, a strong genetic association between specific polymorphisms in the TCF7L2 (TCF4) gene, an effector of the Wnt pathway, and diabetes has been described (), suggesting that Wnt/β-catenin may represent a link between diabetes and cancer.

High insulin levels, as an adaptation to insulin resistance at the onset of diabetes or as a result of exogenous administration, may promote cell growth and cancer by acting through the insulin growth factor receptor (IGFR) family (). However, hyperinsulinemia appears to be secondary to hyperglycemia and might not explain the increased cancer risk in the nondiabetic obese or hyperglycemic population. An alternative possibility is that high serum glucose levels may directly modulate cancer-related signaling pathways, especially given the increased-glucose-consumption characteristic of cancer cells ().

“The dose makes the poison,” wrote Paracelsus in the 16century, and this is especially relevant for diabetics in reference to glucose. Less known, although widely accepted, is the fact that certain cancers are found with an increased frequency in the obese and/or diabetic population. Although it is increasingly important and strongly supported by epidemiological evidence (), little is known of the mechanistic origins of the diabetes- and obesity-cancer link.

Nuclear β-catenin is a well-known marker of malignancy in a wide range of cancers. High glucose consumption directed toward glycolysis is the metabolic hallmark of cancer cells. We reasoned that high glucose uptake by cells could contribute to the tumor phenotype by promoting nuclear accumulation of β-catenin to activate proliferation-related genes. We assayed several tumor-derived cell lines to determine whether their intranuclear β-catenin accumulation was dependent on glucose ( Figure 6 A). Our data revealed that in the presence of LiCl, all tumor-derived cell lines tested, including enteroendocrine (STC-1), colon (HT-29), pancreas (AsPC-1), ovary (OVCAR3), and breast (MDA-MB-231), respond to high glucose by accumulating β-catenin in the nucleus. We then analyzed whether increased LEF-1 and β-catenin binding to the cyclin D promoter was dependent on glucose. Figure 6 B shows a representative semiquantitative PCR on chromatin immunoprecipitates from HT-29 cells; Figure 6 C shows statistical analysis of qPCR on chromatin immunoprecipitated from three biological replicates. Increased binding of both LEF-1 and β-catenin at the cyclin D promoter follows combined treatment with glucose and LiCl in HT-29 colon cancer cells. Ovarian cancer cell lines have also been examined with the same result (A.C.-C., J.M.G.-M., and C.G.-J., unpublished results). Thus, high glucose amplifies Wnt signaling by ensuring high intranuclear LEF-1/β-catenin accumulation and binding to its target promoters.

(B and C) Chromatin was immunoprecipitated from HT-29 colon cancer cells. ChIP was followed by semiquantitative PCR (B) or qPCR (C) as in Figure 5 . Normalized values are presented as mean ± SEM (n = 3) on a log2 scale.

The in vivo effects of glucose on the endogenous GIP promoter, as well as on other genes ( Figures 5 E and 5F), were analyzed by chromatin immunoprecipitation (ChIP) followed by PCR. Representative semiquantitative PCRs on immunoprecipitates using LEF-1 and β-catenin antibodies are shown for the GIP promoter ( Figure 5 E, top); the lower panel corresponds to a control intronic sequence of GIP without TCF/LEF elements, and the bottom panel shows a statistical analysis of qPCRs for GIP performed on immunoprecipitates from five biological replicates. Figure 5 F shows similar results for the promoters of other candidate genes regulated by glucose, namely the proglucagon gene GCG that encodes another incretin, GLP-1, and a critical proliferation regulator, cyclin D, where GAPDH was used as a control non-Wnt-regulated gene. The qPCR data (bottom) represents a statistical analysis of qPCRs for GCG or cyclin performed on immunoprecipitates from three biological replicates. At the GIP, GCG, and cyclin D promoters, both LEF-1 and β-catenin were poorly bound in the absence of LiCl. Single treatment with glucose or LiCl alone induced only minor increases in the binding of both LEF-1 and β-catenin. By contrast, in the presence of LiCl, glucose strongly enhanced their binding to these promoters. Results are presented on a log2 scale. Taken together, upon LiCl treatment, glucose increased the binding of LEF-1 and especially that of β-catenin on different Wnt target genes.

The impact of acetylation on the transcriptional functionality of Wnt effectors was then analyzed. TOPFlash or FOPFlash reporters were transfected into STC-1 cells cultured in the presence of LiCl, and the acetylation balance was manipulated with glucose, NAA, and/or RES ( Figure 4 F). In LiCl-stimulated cells, sirtuin inhibition mimics transcriptional induction by glucose through the LEF/Tcf elements present in the TOPFlash reporter, whereas RES effectively blocks the glucose-driven induction. The results indicate that inhibition of sirtuins by the combination of glucose and LiCl enhances Wnt-dependent transcriptional activation and suggest that similar mechanisms may alter transcription from the natural GIP promoter that bears a Wnt-responsive element. Figure 4 G shows that in the presence of LiCl, NAA mimics glucose induction of the GIP promoter and RES blocks the glucose-dependent induction. These results are consistent with transcriptional activity of Wnt effectors being induced via glucose-driven acetylation of β-catenin by p300 and the inhibition of sirtuins. The Δ19 β-catenin mutant that is unresponsive to the glucose and LiCl combination lacks one lysine, K354. Significantly, the K354R β-catenin mutant expressed in cells treated with glucose and LiCl was unable to interact with GST-LEF-1 ( Figure S5 C) and when immunoprecipitated did not interact with p300 ( Figure S5 D; compare with Δ19 and WT β-catenin in both cases). Consequently, the K354R β-catenin mutant exhibited reduced acetylation in response to glucose and LiCl ( Figure 5 A) or after p300 overexpression ( Figure 5 B), was not retained in the nucleus ( Figures 5 A and 5C) despite its cytosolic accumulation (data not shown), and failed to induce GIP transcription upon treatment with glucose and LiCl ( Figure 5 D). Mutant K345R β-catenin previously identified as being acetylated by p300 but with little effect on transcription () served as the control. Targeting of β-catenin K354 by p300 and SIRT1 was confirmed by immunoprecipitations with anti-acetyllysine ( Figure S5 E) and detection of c-myc-tagged β-catenin, or vice versa ( Figure S5 F). WT β-catenin, but not K354R mutant β-catenin, was acetylated by p300 in cells cultured with LiCl as shown previously; acetylation was enhanced by NAA inhibition of SIRT activity and was blocked by inhibition of p300 with C646 or overexpression of SIRT1. Thus, K354 plays a critical and highly specific role in promoting β-catenin nuclear accumulation and transcriptional activation in response to LiCl and glucose.

(F) ChIP at the proglucagon (GCG), cyclin D, and GAPDH promoters as indicated. GAPDH is used as a control gene not regulated by Wnt. Preimmune serum and ChIP from input chromatin are shown as controls. For qPCRs, normalized values were calculated as fold induction of samples treated with LiCl in the presence versus absence of glucose and presented as mean ± SEM (n = 5) on a log2 scale. See also Figure S5

(D) Cells as in (A) were cotransfected with GIP-luciferase reporter. Relative luciferase units (RLU) were calculated as in Figure 4 G. The western blot shown is representative and shows the expression levels of the mutants.

(A–C) Nuclear extracts (NE) of STC-1 cell transfected with myc-tagged versions of β-catenin: WT, Δ19, or K354R mutants cultured with LiCl and treated as indicated analyzed for β-catenin nuclear accumulation; (A) and (C) show the analysis in crude nuclear extracts, and (B) in immunoprecipitates of the NE using c-myc antibody after cotransfection with Flag-tagged p300 expression vector.

Overexpression of Flag-p300 upon LiCl-induced β-catenin accumulation increased β-catenin nuclear accumulation as much as glucose did ( Figure 4 A). Conversely, C646, a specific inhibitor of p300 acetyltransferase activity, abolished glucose-induced nuclear accumulation and also acetylation upon LiCl treatment ( Figure 4 B), indicating that p300 interaction and activity is a limiting step required for β-catenin nuclear accumulation.

(A–C, E) Fractionated extracts: Nuclear (NE) and cytoplasmic extracts (CE) from STC-1 cells cultured with LiCl and treated as indicated were analyzed by western blotting for β-catenin nuclear accumulation. Erk2 is the loading control. Values represent mean ± SEM (n ≥ 3).

To test whether β-catenin acetylation is increased under the conditions required for nuclear accumulation, we immunoprecipitated nuclear extracts with acetyllysine antibody and detected β-catenin ( Figure 3 G, upper panels) or immunoprecipitated with anti-β-catenin and detected anti-acetyllysine ( Figure 3 G, lower panels). In both cases, acetylated β-catenin was increased in the nucleus of cells cultured under combined glucose and LiCl treatment, conditions wherein p300 interaction is favored and SIRT1 interaction is decreased.

The requirement of LEF-1 for mediating the p300/β-catenin interaction was examined in a western blot for p300 on anti-myc immunoprecipitates from cells transfected with Myc-tagged wild-type (WT) or Δ19β-catenin mutant and treated with LiCl, glucose, both, or none ( Figure 3 F). Whereas ectoptic WT β-catenin bound p300 upon exposure of the cells to both glucose and LiCl (like endogenous β-catenin), conditions that promote LEF-1 and β-catenin interaction and nuclear accumulation of β-catenin, the Δ19 β-catenin mutant was unable to bind p300 under any conditions. Given that p300 binds the C terminus of β-catenin () more than 300 amino acids away from the region missing in Δ19 β-catenin (arms 5–6), our results suggest that LEF-1/β-catenin complexes recruit p300 more efficiently than does β-catenin alone. Thus, the LEF-1 and β-catenin interaction promoted by the combination of glucose and LiCl appears critical for stabilizing p300 interaction and nuclear retention.

Endogenous β-catenin underwent nuclear accumulation following glucose and LiCl stimulation; by contrast, a β-catenin mutant unable to interact with LEF-1 (Δ19 β-catenin) () was unable to accumulate in the nucleus ( Figure 3 E), suggesting that β-catenin/LEF-1 interaction was required for nuclear retention.

An in vivo structure-function study of armadillo, the beta-catenin homologue, reveals both separate and overlapping regions of the protein required for cell adhesion and for wingless signaling.

Immunoprecipitates of nuclear β-catenin were then examined. Strikingly, β-catenin and p300 interaction was detected only in cells treated with both glucose and LiCl and not in control cells or cells with a single glucose or LiCl treatment ( Figure 3 C). SIRT1 and β-catenin interactions increased with glucose alone more than 2-fold, but LiCl disrupted SIRT1 interactions ( Figure 3 D). Collectively, the results indicate that combined glucose and LiCl treatment increases p300-LEF-1-β-catenin interaction while preventing SIRT1-LEF-1 and SIRT1-β-catenin interaction and suggest that increased acetylation by p300 may be required for nuclear retention of the LEF-1/β-catenin complex.

Given that both p300 and SIRT1 activities control the localization of other transcription factors, their interactions with LEF-1 were examined in anti-LEF-1 immunoprecipitates ( Figure 3 A). Glucose and LiCl cooperated to increase the level of p300/LEF-1 complexes by 3.5-fold, consistent with results shown in Figure 2 E, whereas single treatment did not have a significant influence. By contrast, SIRT1/LEF-1 complexes were increased 2.5-fold with glucose alone compared to cells untreated or treated with LiCl alone ( Figure 3 B). Strikingly, LiCl disrupted the SIRT1/LEF-1 complexes induced by glucose, most likely owing to competition for LEF-1 binding between β-catenin (accumulated by LiCl) and SIRT1. Glutathione S-transferase (GST)-LEF-1 pull-down confirmed the immunoprecipitation results ( Figures S5 A and S5B) obtained using LEF-1 antibodies. Thus, upon LiCl treatment, glucose promotes the formation and nuclear retention of LEF-1 complexes with β-catenin and p300, but not with SIRT1.

(A–D, F, and G) Immunoprecipitation of STC-1 nuclear extracts with the antibodies indicated at the bottom (e.g., IP LEF-1 in A). Input (10%) and Erk2 in flow through (as loading control) are shown.

Representative immunoblots (IB) with the antibodies indicated on the left of each panel and quantification; values represent mean ± SEM, n = 3 in all panels.

Given that acetylation of β-catenin through p300 or CBP () and its deacetylation by SIRT1 () impacts its oncogenicity, we examined whether combined LiCl and glucose treatment altered the levels of the acetylase p300 and the deacetylase SIRT1. Importantly, combined LiCl and glucose additively increased p300 levels ( Figure 2 E), and although SIRT1 levels were unaffected ( Figure 2 F), combined LiCl and glucose cooperated to reduce sirtuin activity by 40% ( Figure 2 G). Glucose or LiCl alone did not reduce sirtuin activity (data not shown). Upon LiCl treatment, the effect of glucose on sirtuin activity was largely abolished by the sirtuin enhancer resveratrol (RES) and was reproduced by the sirtuin inhibitor nicotinamide (NAA). Together, these results suggest that combined glucose and LiCl treatments cooperate to profoundly alter the acetylation balance by increasing levels of the acetylase p300 and reducing the deacetylase activity of sirtuins.

β-catenin is unable to bind directly to DNA but activates incretin expression through an element that binds Wnt effectors such as LEF-1. Combined glucose and LiCl do not increase LEF-1 expression (J.M.G.-M. and C.G.-J., unpublished data) but lead to nuclear accumulation of β-catenin, despite lacking a classical nuclear localization signal (NLS). Because LEF-1 possesses its own NLS () and is able to interact with β-catenin, we asked whether glucose favored LEF-1 and β-catenin interaction as a possible mechanism for the nuclear accumulation of β-catenin. Immunoprecipitation experiments indicated that LiCl and glucose cooperate to selectively favor nuclear LEF-1 and β-catenin interaction, and neither glucose nor LiCl alone allowed complex formation ( Figure 2 D). Overexpression of LEF-1 in LiCl-stimulated cells induced nuclear accumulation of β-catenin, even in the absence of glucose ( Figure S4 A), and increased GIP-promoter activity ( Figure S4 B), whereas a dominant negative LEF-1 (DN-LEF-1) unable to bind β-catenin blocked the induction of GIP transcription by LiCl ( Figure S4 C) or β-catenin ( Figure S4 D). Thus, the availability of LEF-1 may be critical for LEF-1 and β-catenin interaction and nuclear accumulation.

Taken together, the results thus far indicate that neither Wnt or LiCl alone nor high glucose alone can trigger nuclear accumulation of β-catenin. However, high glucose will promote nuclear accumulation of β-catenin and activation of its target promoters if β-catenin is stabilized by prior inactivation of GSK3β with Wnt-3a or LiCl.

Importantly, glucose-induced nuclear accumulation of β-catenin ( Figure S3 A) occurred between 1 hr and 4 hr, matched the time required for transcriptional induction as shown in Figure 1 E, and was specific to high glucose, in that using glutamine as an alternative carbon source did not substitute for glucose in promoting nuclear accumulation of β-catenin ( Figure S3 C). Glucose targeted β-catenin specifically and did not alter the control of other transcription factors by growth factors, such as FoxO1 regulation by Insulin or Smads regulation by transforming growth factor β (TGFβ) (data not shown).

Thus, LiCl or Wnt-3a inhibits GSK3β, inducing cytosolic accumulation of β-catenin, which allows high glucose to induce nuclear β-catenin accumulation, and neither cytosolic nor nuclear β-catenin accumulation relies on Akt activation.

Both LiCl and Wnt-3a induce Ser9 phosphorylation of GSK3β to inhibit GSK3β-driven β-catenin degradation and lead to β-catenin cytosolic accumulation (time courses, Figures S2 B and S2C). However, nuclear accumulation of β-catenin required the addition of high glucose for at least 1 hr and remained up to 24 hr ( Figure 2 C). Importantly, alternative GSK3β inhibitors, BIO and SB 216763 instead of Wnt or LiCl, also cooperated with glucose to induce β-catenin nuclear accumulation ( Figure S2 D) and transcriptional activity ( Figure S2 E). Consistently, expression of a kinase-inactive (KI) mutant of GSK3β also led to β-catenin-activated transcription in cells cultured with high glucose ( Figure S2 F). Neither LiCl nor high-glucose effects were mediated through altered Akt phosphorylation ( Figure S2 G), compared with insulin used as positive control. Moreover, glucose alone did not inactivate GSK3β, because neither pGSK3β (Ser 9) nor β-catenin levels were increased unless LiCl was also present at any time or dosage ( Figures S3 A–S3B).

Western blotting of fractionated cell extracts ( Figure 2 B) confirmed that LiCl alone induced elevated cytoplasmic β-catenin, but nuclear accumulation of β-catenin required both glucose and LiCl ( Figure 2 B). Lamin B1 and GAPDH are shown as nuclear and cytoplasmic fractionation controls, respectively, and total ERK is shown as the loading control. Only 25 mM of glucose (not 5 mM) triggered nuclear accumulation of β-catenin in the presence of LiCl ( Figure S2 A), consistent with the transcriptional induction of the TOPFlash and GIP reporters ( Figures 1 F and 1G). Thus, LiCl and Wnt-3a induce cytosolic β-catenin accumulation, but nuclear accumulation requires high glucose in addition.

Given that Wnt-3a (or LiCl) induces GIP promoter activity and cytosolic accumulation of the strong transcriptional coactivator β-catenin, confocal microscopy was used to study whether glucose regulates β-catenin subcellular distribution. The results ( Figure 2 A) indicate that β-catenin staining was restricted to focal adhesions in cells cultured in the absence of LiCl or Wnt and glucose (control) and that glucose alone did not alter the location of β-catenin. Lamin B (red) staining delimits the nuclear membrane. Wnt-3a or LiCl alone displaced β-catenin from the cell periphery and increased β-catenin accumulation in the cytoplasm, but not in the nucleus. Strikingly, addition of glucose (25 mM) to cells cultured with Wnt-3a resulted in substantial nuclear accumulation of β-catenin. Nuclear accumulation of β-catenin occurred early, between 1 hr and 4 hr after glucose addition, and was reproduced upon the addition of glucose to cells treated with LiCl, although the proportion of β-catenin retained in the cytoplasm was higher than with Wnt-3a.

(G) NAD-dependent deacetylase activity in STC-1 cells cultured with sirtuin inhibitor NAA (300 μM) or enhancer RES (50 μM). Relative luciferase units (RLU) was calculated as fold induction relative to the corresponding control. See also Figures S2– S5

Because LiCl induces GIP expression in enteroendocrine cells through stabilization of the Wnt effector β-catenin that is targeted to the proximal GIP promoter (), we asked whether glucose also acted through this natural Wnt-responsive element. To this end, we transfected a series of GIP promoter deletion mutants and challenged them with glucose. Deletion of the promoter region containing the previously reported Wnt-dependent TL5 element abrogated the response to glucose ( Figure 1 I). Mutagenesis of the TL5 element confirmed that its integrity is required for glucose induction ( Figure 1 J). Thus, high glucose enhances LiCl- or Wnt-dependent GIP expression through molecules that bind the Wnt-dependent TL5 element. Significantly, the effects of the WNT glucose combination were not restricted to the GIP promoter, but were also observed on TOPFlash and other WNT-responsive genes (see also below).

If glucose regulates the Wnt signaling pathway, it should alter the responsiveness of a consensus Wnt-dependent element. TOPFlash, bearing eight copies of the consensus LEF/Tcf binding site or the mutated FOPFlash reporters (), was transfected into STC-1 cells. LiCl modestly increased the TOP/FOP ratio 2.5-fold ( Figure 1 D), whereas glucose alone was unable to activate this promoter. However, glucose substantially amplified the effect of LiCl, increasing the TOP/FOP ratio up to 8-fold. Transcriptional synergy between glucose and LiCl was significant between 1 and 4 hr and increased up to 24 hr ( Figure 1 E). Importantly, glucose was also required for activation of the GIP promoter by LiCl or Wnt ( Figures S1 A and S1B available online), and the synergy was independent of the order of addition. Importantly, only concentrations that mimic hyperglycemia (a glucose of 25 mM and not 5 mM) synergized with LiCl for activation of the TOPFlash ( Figure 1 F) or GIP ( Figure 1 G) reporters. Because both concentrations of glucose increased ATP levels to a similar extent ( Figure 1 H) and independently of the presence of LiCl ( Figures S1 C and S1D), the results suggest that the transcriptional synergy observed with glucose and LiCl or Wnt is specific to high glucose and is not accounted for by increased ATP production.

Glucose is the physiological stimulus for GIP secretion, but whether glucose induces GIP transcription is not known. The effect of glucose depletion on basal GIP expression was examined in enteroendocrine STC-1 cells transfected with a GIP promoter-luciferase reporter () ( Figure 1 A). Glucose deprivation for 24 hr or 48 hr led to 50% and 80% reduction in basal GIP promoter activity, respectively. The effect of glucose addition on GIP expression was then evaluated and compared to the effect of lithium chloride (LiCl), which mimics Wnt-3a signaling in STC-1 cells to induce GIP expression through a LEF/Tcf element (). Surprisingly, neither LiCl nor glucose alone induced the GIP promoter, but in the presence of LiCl, glucose induced a 2.2-fold increase in GIP-promoter activity ( Figure 1 B). Likewise, combined glucose and LiCl increased endogenous GIP messenger RNA (mRNA) by 8-fold ( Figure 1 C), whereas either stimulus alone promoted a very modest induction of endogenous GIP, as determined using quantitative RT-PCR (qRT-PCR). These results suggest that glucose targets molecules regulated by Wnt or LiCl and explain why induction of GIP by glucose has previously been so difficult to observe in enteroendocrine STC-1 cells.

(I and J) Glucose induction of GIP-luciferase reporter on mutants: deletions (I), point mutation at Wnt-responsive TL5 element (J). Relative luciferase units (RLU) was calculated as fold induction relative to the corresponding control in all experiments. Values represent mean ± SEM; n ≥ 3. Statistical analysis in all the work was via ANOVA, n ≥ 3 unless otherwise stated. See also Figure S1

(C) qRT-PCR of GIP mRNA in cells treated as in (B). Values normalized with endogenous control (18S) are referred to as fold induction over untreated cells.

(B) Cells deprived of glucose for 36 hr were stimulated with glucose and/or LiCl. Glucose concentration was 25 mM in all experiments unless otherwise indicated, and LiCl was 20 mM.

Discussion

Kim and Hay, 2001 Kim K.

Hay E.D. New evidence that nuclear import of endogenous beta-catenin is LEF-1 dependent, while LEF-1 independent import of exogenous beta-catenin leads to nuclear abnormalities. Hecht et al., 2000 Hecht A.

Vleminckx K.

Stemmler M.P.

van Roy F.

Kemler R. The p300/CBP acetyltransferases function as transcriptional coactivators of beta-catenin in vertebrates. Lévy et al., 2004 Lévy L.

Wei Y.

Labalette C.

Wu Y.

Renard C.A.

Buendia M.A.

Neuveut C. Acetylation of beta-catenin by p300 regulates beta-catenin-Tcf4 interaction. Wolf et al., 2002 Wolf D.

Rodova M.

Miska E.A.

Calvet J.P.

Kouzarides T. Acetylation of beta-catenin by CREB-binding protein (CBP). Ma et al., 2005 Ma H.

Nguyen C.

Lee K.S.

Kahn M. Differential roles for the coactivators CBP and p300 on TCF/beta-catenin-mediated survivin gene expression. von Kries et al., 2000 von Kries J.P.

Winbeck G.

Asbrand C.

Schwarz-Romond T.

Sochnikova N.

Dell’Oro A.

Behrens J.

Birchmeier W. Hot spots in beta-catenin for interactions with LEF-1, conductin and APC. Despite the importance of Wnt/β-catenin signaling for development, tissue renewal, and cancer, the mechanisms by which β-catenin enters and is retained in the nucleus under Wnt stimulation are poorly understood. Given that β-catenin lacks a classical NLS, its nuclear entry is likely to depend on its interactions with other molecules, such as LEF-1, with their own NLS. Although we and others () have observed that overexpression of LEF-1 results in nuclear entry of β-catenin, the physiological stimulus for nuclear accumulation of β-catenin has not previously been identified. That acetylation might be implicated was indicated by studies in which overexpressed p300 or CBP could interact with the C terminus of β-catenin (), leading to lysine acetylation, binding to TCF/LEF factors (), nuclear entry of β-catenin (), and increased oncogenic potential (). However, mutation of the acetylated residue identified, K345, did not affect β-catenin-driven transcription activation. Our results indicate that the physiological trigger underlying nuclear accumulation of β-catenin in cooperation with Wnt or LiCl is high glucose. In the presence of LiCl, glucose performs two complementary functions directed toward enhancing nuclear accumulation of acetylated β-catenin. First, it increases p300 expression and enhances its interactions with LEF-1 and β-catenin; second, it inhibits sirtuin deacetylase activity. Together, these events lead to increased β-catenin acetylation and nuclear accumulation. Moreover, we identify K354 as an essential residue for glucose induction of β-catenin nuclear accumulation. K354 lies within the region absent in the Δ19 mutant, a region previously identified as being critical for β-catenin interaction with LEF-1 (). The effects are highly specific, because glucose cannot be substituted by another carbon source such as glutamine and the effects of other growth factors such as Insulin or TGFβ are independent of glucose (data not shown).

Collectively, the data presented are consistent with a model in which p300/CBP acetylation of lysine residues in the armadillo repeats increase binding of TCF/LEF, leading to β-catenin nuclear accumulation. In this respect it is possible that β-catenin may treadmill between the nucleus and the cytoplasm and that its recruitment to DNA via its acetylation-enhanced interaction with LEF-1 would lead to its effective nuclear retention. Our results are also consistent with LEF-1 promoting interaction of β-catenin with p300, thereby facilitating the probable formation of a tripartite nuclear complex that would mediate gene regulation.

Interestingly, SIRT1 interaction with both β-catenin and LEF-1 is increased in response to high glucose, but these complexes are disrupted in the presence of LiCl. Although the precise molecular mechanisms have not been explored here, we view it as probable that SIRT1 acts as a competitor for interaction between LEF-1 and β-catenin. Such a scenario would be advantageous, because it would ensure that β-catenin nuclear accumulation would only occur on receipt of a simultaneous Wnt and high-glucose signal; the low level of β-catenin observed in the absence of a Wnt signal would be insufficient to activate gene expression even in the presence of glucose, because it would not be able to displace SIRT1 from LEF-1.

Firestein et al., 2008 Firestein R.

Blander G.

Michan S.

Oberdoerffer P.

Ogino S.

Campbell J.

Bhimavarapu A.

Luikenhuis S.

de Cabo R.

Fuchs C.

et al. The SIRT1 deacetylase suppresses intestinal tumorigenesis and colon cancer growth. Audrito et al., 2011 Audrito V.

Vaisitti T.

Rossi D.

Gottardi D.

D’Arena G.

Laurenti L.

Gaidano G.

Malavasi F.

Deaglio S. Nicotinamide blocks proliferation and induces apoptosis of chronic lymphocytic leukemia cells through activation of the p53/miR-34a/SIRT1 tumor suppressor network. Sauve and Schramm, 2003 Sauve A.A.

Schramm V.L. Sir2 regulation by nicotinamide results from switching between base exchange and deacetylation chemistry. + obtained by lactic acid production. Thus, high glucose may promote the depletion of NAD+, thereby limiting the amount of this essential sirtuin cofactor and consequently the deacetylase activity of sirtuins. Although SIRT1 may play a role via stable and direct binding to LEF-1 or β-catenin, it is also clear that its catalytic activity is particularly important. SIRT1 deacetylates β-catenin to decrease its oncogenic capacity (), and SIRT1 depletion, or inhibition using NAA, in cells treated with LiCl mimics glucose-driven nuclear retention of β-catenin for promoting transcriptional activation. Thus, the effect of glucose is likely to be mediated, at least partially, via the inhibition of sirtuins. The reduction in sirtuin activity obtained in cells treated with NAA or with glucose and LiCl is greater than that obtained in other cells with NAA at doses 10- to 30-fold higher (), although it is understandably less than the inhibition obtained using NAA in vitro under chemically defined conditions with purified protein (). Because tumor cells exhibit enhanced glycolysis, obtaining much lower energy per molecule of glucose than normal cells through respiration, under LiCl stimulation high glucose uptake may speed up glycolysis, leading to rapid turnover of the NADobtained by lactic acid production. Thus, high glucose may promote the depletion of NAD, thereby limiting the amount of this essential sirtuin cofactor and consequently the deacetylase activity of sirtuins.