Pancreatic β-cells are highly responsive to changes in glucose, but the mechanisms involved are only partially understood. There is increasing evidence that the β-catenin signalling pathway plays an important role in regulating β-cell function, but the mechanisms regulating β-catenin signalling in these cells is not well understood. In the present study we show that β-catenin levels and downstream signalling are regulated by changes in glucose levels in INS-1E and β-TC6-F7 β-cell models. We found a glucose-dependent increase in levels of β-catenin in the cytoplasm and nucleus of INS-1E cells. Expression of cyclin D1 also increased with glucose and required the presence of β-catenin. This was associated with an increase in phosphorylation of β-catenin on Ser552, which is known to stabilize the molecule and increase its transcriptional activity. In a search for possible signalling intermediates we found forskolin and cell-permeable cAMP analogues recapitulated the glucose effects, suggesting a role for cAMP and PKA (cAMP-dependent protein kinase/protein kinase A) downstream of glucose. Furthermore, glucose caused sustained increases in cAMP. Two different inhibitors of adenylate cyclase and PKA signalling blocked the effects of glucose, whereas siRNA (small interfering RNA) knockdown of PKA blocked the effects of glucose on β-catenin signalling. Finally, reducing β-catenin levels with either siRNA or pyrvinium impaired glucose- and KCl-stimulated insulin secretion. Taken together the results of the present study define a pathway by which changes in glucose levels can regulate β-catenin using a mechanism which involves cAMP production and the activation of PKA. This identifies a pathway that may be important in glucose-dependent regulation of gene expression and insulin secretion in β-cells.
- cAMP-dependent protein kinase/protein kinase A (PKA)
- cyclin D1
Changes in blood glucose levels stimulate a wide range of responses in pancreatic β-cells and these ultimately play a key role in regulating glucose metabolism. These responses include insulin secretion  and insulin gene expression , as well as pathways controlling cell growth, cell death and cell division [3–6]. A number of different mechanisms have been implicated in glucose sensing in β-cells [2,7–9], and glucose-induced autocrine insulin signalling can also contribute to gene expression .
We have previously shown that the β-catenin signalling pathway can be regulated by glucose in some cell types ; however, this has not been investigated in β-cells, another cell type known to respond to glucose. The potential importance of this pathway in β-cells has been highlighted by the finding that single nucleotide polymorphisms in the TCF7L2 (transcription factor 7-like 2) gene are a major risk factor for the development of Type 2 diabetes [12–14]. Evidence indicates that TCF7L2 polymorphisms are associated with detrimental effects on β-cell function [15,16], including β-cell survival and glucose-stimulated insulin secretion [17–20]. TCF7L2 is a β-catenin transcriptional partner protein and previous studies have found that the β-catenin signalling pathway has been implicated in several processes involved in regulating metabolism, including in β-cells . The inference from this has been that defective β-catenin signalling may play an important role in regulating β-cell function.
The cytosolic pool of β-catenin is normally held in a tight complex with GSK3 (glycogen synthase kinase 3)–Axin–APC (adenomatous polyposis coli)–CK1 (casein kinase 1) allowing constitutive phosphorylation of β-catenin on its N-terminal domain which targets it for proteasomal degradation . The β-catenin signalling pathway is activated when the cytosolic pool of β-catenin is stabilized and thus able to move to the nucleus where it can bind to and activate the transcription factor TCF7L2, inducing expression of genes such as AXIN2 or cyclin D1 (HGNC symbol CCND1) . The best understood mechanism for activating the pathway involves Wnt-mediated dissociation of the GSK3–Axin–APC–CK1 complex resulting in reduced phosphorylation of β-catenin on the N-terminus, and hence an accumulation of this in the cytoplasm. However, evidence has indicated that the stability and transcriptional activity of β-catenin can be further modulated by phosphorylation of its C-terminal region, at Ser552 and Ser675 [24–26]. This provides the opportunity for the canonical Wnt signalling pathway to cross-talk with other signalling pathways.
In the present study, we have investigated whether glucose can also regulate β-catenin and its downstream signalling pathways in β-cell models. We found that the level of β-catenin and transcription of its target gene, CCND1, increase in a glucose-dependent manner, and this is due to a glucose-induced increase in cAMP levels and a subsequent activation of PKA (cAMP-dependent protein kinase/protein kinase A) and phosphorylation of β-catenin on Ser552. We show that the changes in β-catenin levels can have an impact on GSIS (glucose-stimulated insulin secretion). The findings of the present study identify a new mechanism by which β-cells can regulate important cellular processes in response to changes in glucose concentration.
INS-1E β-cells (provided by Professor CB Wollheim ) were maintained in RPMI 1640 medium supplemented with 10% (v/v) FBS (fetal bovine serum), 100 units/ml penicillin, 100 μg/ml streptomycin (Gibco, Life Technologies), 10 mM Hepes, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol (Sigma–Aldrich). Experiments were performed on 80% confluent cells after a 4 h serum-starvation in RPMI 1640 medium without glucose. Then the medium was renewed and glucose (10 mM) and other treatments were added as indicated. Somatostatin, H-89, forskolin, ddAdo (2′,3′-dideoxyadenosine), IBMX (isobutylmethylxanthine), AICAR (5-amino-4-imidazolecarboxamide riboside), db-AMP (dibutyryl-cAMP) and pyrvinium pamoate were from Sigma–Aldrich. Octreotide was from Novartis Pharmaceuticals. The β-TC cell line was kindly provided by Dr Shimon Efrat (Tel Aviv University, Ranat Aviv, Israel).
siRNA (small interfering RNA) experiments
All of the reagents for siRNA experiments were purchased from Invitrogen Life Technologies. To knock down β-catenin or PKA, we transfected INS-1E cells with specific siRNA (50 pM) or with the negative Universal Control™ (scrambled control) using Lipofectamine™ 2000 transfection reagent.
Western blot analysis
Western blot analysis was carried out using antibodies against active β-catenin (i.e. non-phosphorylated β-catenin Ser33/Ser37/Thr41) or total β-catenin (Symansis), α-tubulin and β-actin (Sigma–Aldrich). All other antibodies were from Cell Signaling Technology. Subcellular fractionation was performed using the NE-PER kit (Pierce, Thermo Fisher Scientific).
qRT-PCR (quantitative real-time-PCR) analysis
All of the reagents for qRT-PCR were purchased from Invitrogen Life Technologies. RNAs were extracted using TRIzol® or with the PureLink™ Micro-to-Midi™ Total RNA Purification system after siRNA transfection. We used the SuperScript® III First-Strand Synthesis system for cDNA synthesis. Real-time PCR were performed using an Applied Biosystems 7900 instrument and software using SYBR Green mastermix with ROX®. Primer sequences are available from the corresponding author on request.
INS-1E cells were plated in 96-well plates and IBMX was added during treatments. cAMP concentrations were measured using the Alpha-Screen cAMP assay from PerkinElmer.
Measurement of insulin concentration
INS-1E cells were starved in KRBH (Krebs-Ringer Bicarbonate Hepes) buffer (119 mM NaCl, 4.74 mM KCl, 1.19 mM MgSO4, 25 mM NaHCO3, 1.19 mM KH2PO4, 2.54 mM CaCl2 and 50 mM Hepes), pH 7.4, with 1% BSA for 1 h, stimulated in the same buffer as indicated for 2 h, and media were collected. The insulin concentration was measured using the AlphaLISA Insulin Assay kit from PerkinElmer.
Results are presented as means±S.E.M. with the number of experiments indicated in the Figure legends. Statistical differences were determined by Student's t test or by one-way ANOVA followed by Tukey's test or Dunnett's test as indicated. Statistical significance is displayed as *P<0.05 or **P<0.01.
Glucose regulates β-catenin levels in INS1-E β-cells
Levels of both active (i.e. not phosphorylated at the N-terminus Ser33/Ser37/Thr41 sites) and total β-catenin were significantly higher after glucose-starved INS-1E β-cells were stimulated for up to 4 h with 10 mM glucose (2.8- and 1.8-fold increases respectively; Figure 1A). We observed that the total β-catenin protein levels were higher with glucose in both the cytoplasm and nucleus (Figure 1B), with a greater increase in the nucleus (2.6-fold compared with 1.4-fold). Moreover, we found that β-catenin levels increased in a dose-dependent manner (Figure 1C) and reached a maximum with 5 and 10 mM glucose. We chose to use a glucose concentration of 10 mM for all experiments, as the results were more consistent at this concentration. We also observed that β-catenin levels increased in a time-dependent manner (Supplementary Figure S1 at http://www.BiochemJ.org/bj/449/bj4490803add.htm).
Glucose regulates cyclin D1 levels via β-catenin in INS-1E β-cells
In addition to the changes in β-catenin, we found that mRNA levels for the β-catenin target gene cyclin D1 were 2-fold higher following glucose treatment (Figure 2A). Cyclin D1 mRNA levels increased with glucose in a time-dependent way between 2 and 4 h, and then started decreasing at 6 h. The fold increase was at a maximum at 4 h (2.1-fold), compared with 2 (1.5-fold), 3 (1.8-fold) and 6 (1.6-fold) h (Supplementary Figure S2 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). Levels of cyclin D1 protein were also higher with 10 mM glucose (1.8-fold increase compared with the control; Figure 2B). Since glucose stimulation of INS-1E β-cells results in insulin secretion, we used two approaches to investigate whether the effects of glucose on β-catenin signalling were caused by autocrine actions of insulin. However, neither somatostatin nor octreotide blocked the response to glucose, and adding exogenous insulin did not mimic the glucose effect (Supplementary Figure S3 at http://www.BiochemJ.org/bj/449/bj4490803add.htm).
β-Catenin is only one of many pathways that have the potential to regulate cyclin D1 levels , so to test what contribution β-catenin made to glucose regulation of cyclin D1 in β-cells, we suppressed β-catenin expression using siRNA. The siRNAs used decreased protein levels of active and total β-catenin down to 3 and 5% of starting levels respectively (Figure 2C). Looking at β-catenin (HGNC symbol CTNNB1) mRNA levels under knock-down conditions, we found that the level was reduced to one-third compared with the control conditions, and was also significantly reduced (Supplementary Figure S4 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). All of the experiments with siRNA were repeated with a second siRNA sequence which gave similar results. Knockdown of β-catenin completely prevented the glucose-induced increase in cyclin D1 mRNA level in INS-1E β-cells (Figure 2D). Pyrvinium is a potent inhibitor of the β-catenin signalling pathway . In INS-1E β-cells, pyrvinium (100 nM) also decreased β-catenin levels (Figure 3A) and inhibited glucose-induced cyclin D1 expression (Figure 3B). Taken together these results indicate that β-catenin is required for the glucose-dependent increase in cyclin D1 expression in INS-1E β-cells.
Glucose-induced increases in β-catenin signalling require PKA
One possible mechanism by which glucose could regulate β-catenin levels is via increasing phosphorylation of GSK3α/β on Ser9/Ser21 as this would reduce GSK3 activity and so reduce phosphorylation of N-terminal sites on β-catenin, resulting in its stabilization. However, glucose did not induce any increase in phosphorylation of GSK3 after a 4 h stimulation (Supplementary Figure S5 at http://www.BiochemJ.org/bj/449/bj4490803add.htm).
β-Catenin is also regulated by phosphorylation at Ser675 [25,29] and Ser552 [24,26,30]. This results in stabilization of the protein and activation of its transcriptional activity [25,29]. Phosphorylation at Ser675 was readily detectable in glucose-starved INS-1E β-cells, but we did not see any significant changes following the addition of glucose (Supplementary Figure S6 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). However, we did observe significant changes in phosphorylation of Ser552 in response to increases in glucose levels (Figure 4A). Importantly it has been shown that canonical Wnt signalling does not affect the phosphorylation of this site . Other possible signalling pathways include AMPK (AMP-activated protein kinase) as it is known to be regulated by changes in glucose levels . However, we did not observe any effect of AICAR, an activator of AMPK, on β-catenin levels or on cyclin D1 expression in INS-1E cells (Supplementary Figure S7 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). Ser552 has also been described as a site for Akt , but we did not observe any increase in β-catenin in insulin-stimulated INS-1E cells (Supplementary Figure S3). Another kinase that has been reported to phosphorylate Ser552 is PKA . If PKA were involved then the cAMP analogues and forskolin would also activate β-catenin signalling in these cells. To test whether cAMP/PKA were involved in the mechanism, we added a cell permeable analogue of cAMP (db-cAMP) to INS-1E β-cells and checked whether it could mimic the glucose effect. First, db-cAMP itself was able to up-regulate the phosphorylation of β-catenin on Ser552 (Figure 4A). We also found that phosphorylation on Ser552 increased in both the cytoplasm and the nucleus in response to glucose and/or db-cAMP (Figure 4B). As a consequence, β-catenin levels also increased, as β-catenin is more stable and can accumulate in the cytoplasm to then translocate into the nucleus. Forskolin also stimulated the phosphorylation of β-catenin on Ser552 in INS-1E β-cells (Supplementary Figure S8 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). As described above, we found that cAMP could increase the phosphorylation of GSK3 but, surprisingly, when glucose was added with cAMP, GSK3 phosphorylation decreased compared with cAMP alone (Supplementary Figure S5). Next we found that cAMP could increase cyclin D1 expression in INS-1E β-cells, and found the cAMP effect was almost completely inhibited when β-catenin was knocked-down (Figure 5). This confirms that cAMP-activated cyclin D1 expression requires β-catenin in INS-1E β-cells.
Taken together the findings of the present study suggested that glucose could be activating β-catenin via an increase in PKA activity. Since this would require a rise in cAMP, we measured cAMP concentrations at different time points after the addition of glucose. We found that changing glucose levels alone was able to induce a sustained increase in cAMP levels in INS-1E β-cells (Figure 6A). Glucose also induced increases in phosphorylation of CREB (cAMP-response-element-binding protein) in a time-dependent manner, which is consistent with activation of PKA by glucose (Supplementary Figure S9 at http://www.BiochemJ.org/bj/449/bj4490803add.htm). To confirm the involvement of PKA in glucose-induced increases in β-catenin, we knocked down PKA-Cα (the catalytic subunit α of PKA) using siRNA. We found that knock down reduced the phosphorylation of β-catenin on Ser552, as well as total β-catenin levels (Figure 6B). We also showed that knocking-down PKA-Cα inhibited the increase in cyclin D1 levels in response to glucose in INS-1E β-cells (Figure 6C), confirming that PKA was required for the glucose effect. In further support of this, the PKA inhibitor H-89 and the adenylate cyclase inhibitor ddAdo also blocked the glucose effect on cyclin D1 levels (Figure 6D).
We found that glucose was also able to increase both active and total levels of β-catenin in another β-cell line (β-TC6-F7) and that pyrvinium could reduce the levels (Figure 7). Glucose and forskolin also increased the phosphorylation of β-catenin on Ser552, corresponding to an increase in the total level of β-catenin (Figure 7).
Finally we looked at the biological effect of increased levels of β-catenin or increased phosphorylation of β-catenin. We found that reducing β-catenin using pyrvinium completely suppressed GSIS in INS-1E β-cells (Figures 8A and 8B). When we knocked-down β-catenin using siRNA, we also observed an alteration of insulin secretion in response to glucose compared with the scrambled control (Figures 8C and 8D). The results of the present study suggest a deregulation of insulin secretion with reduced β-catenin, as the basal insulin secretion is slightly higher compared with the scrambled control and glucose is unable to increase it (Figure 8C). We then looked at insulin secretion in response to KCl. In the presence of glucose, KCl increased insulin secretion, whereas reducing β-catenin with pyrvinium was associated with attenuation of KCl-induced insulin secretion (Figures 8E and 8F). These data show that β-catenin is involved in the mechanisms regulating insulin secretion in β-cells.
The ability of glucose to regulate β-catenin appears to be restricted to a small number of cell types  and the mechanism we have identified in β-cells is clearly different from that in macrophages . We have also investigated whether similar mechanisms might exist in other glucose-responsive cells secreting metabolic hormones, but found no such effect in GluTag cells (L-cell model) or the α-TC1 clone 9 α-cell model (results not shown).
The finding in the present study that changes in glucose levels modify β-catenin levels in a cultured β-cell model is consistent with a previous report indicating that glucose can increase β-catenin levels in human islets , but no mechanism for such increases was reported. The results of the present study provide clear evidence for a pathway in which a glucose-dependent increase in cAMP levels and activation of PKA can result in the activation of the β-catenin signalling pathway in β-cells (Figure 9). This provides the first evidence for a mechanism by which the β-catenin pathway can be used to sense changes in glucose levels in β-cells.
Glucose regulation of cAMP levels in β-cells has been described previously [34–37], with this effect being observed across the range of physiologically relevant glucose levels and being sustained for several hours . This is in agreement with the prolonged activation seen in the present study. However, the potential importance of this glucose-dependent rise in cAMP has been largely overlooked following the finding that the main trigger for glucose-stimulated insulin secretion was production of ATP and a consequent activation of Ca2+ influx into the cell . However, many subsequent studies have shown that hormones such as GLP-1 (glucagon-like peptide-1), which raise cAMP levels, have a powerful potentiating effect on glucose-stimulated insulin secretion , which would suggest that the glucose-induced rise in cAMP could play some similar role.
The glucose-induced increase in cAMP had not previously been linked to an increase in β-catenin, so we investigated the mechanisms that could be causing this. One previous study indicated that, in L-cells, β-catenin stabilization was attributed to PKA-mediated phosphorylation of GSK3 . This inhibitory phosphorylation on Ser9/Ser21 of GSK3 α/β was associated with decreased phosphorylation of the N-terminal region of β-catenin and so stabilization of the molecule . Although we have shown previously that PKA can also phosphorylate and inactivate GSK3 in other tissues , we found in the present study that cAMP could increase phosphorylated GSK3 in INS-1E cells, but by adding glucose with cAMP we reversed this increase. Another possible mechanism is direct phosphorylation of β-catenin by PKA. A previous study found that GLP-1 up-regulates β-catenin in INS-1 β-cells, also via cAMP, and speculated this might be due to phosphorylation of Ser675 of β-catenin . This was because other studies had found that PKA phosphorylates Ser675 and that this stabilizes β-catenin and activates β-catenin transcriptional activity [25,29]. In addition, bioinformatics analysis of the β-catenin sequence using the Scansite algorithm  predicts that Ser675 would be phosphorylated by calmodulin-dependent kinase 2, which would also be activated by glucose via rises in intracellular Ca2+. However, in the present study we show that neither glucose nor pharmacological activators of PKA signalling were able to significantly increase phosphorylation of β-catenin Ser675. This may be because phosphorylation at this site was already maximal or it may be that phosphorylation at this site in vivo requires prior phosphorylation of β-catenin at Tyr654, as has been suggested recently . However, we do observe large increases in Ser552 phosphorylation of β-catenin, and phosphorylation of this site has previously been shown to be a site for PKA phosphorylation , to promote its transcriptional activity [25,26], to be associated with cell proliferation  and to allow it to be stabilized by binding to 14-3-3ζ . Taken together, these results suggest that the glucose- and PKA-induced increase in β-catenin in the nucleus and increased transcription of the cyclin D1 gene is linked to an increased level of phosphorylation on Ser552.
One question raised by the present study is why GSK3 may mediate cAMP signalling to β-catenin in some cell types, whereas direct phosphorylation of β-catenin by PKA occurs in others. This is most likely due to the action of PKA being localized to complexes formed by AKAPs (A-kinase-anchoring proteins) . Indeed AKAP220 will co-localize GSK3 with PKA , whereas β-catenin is held in a complex with PKA via AKAP79/150 . The actual mechanism in β-cells is currently not known, but AKAP79/150 is present in β-cells , suggesting a mechanism for the direct phosphorylation on Ser552 that we observe in the present study.
The glucose-induced increase in β-catenin is likely to play a role in regulating β-cell function. Proliferation could be one of the results of this as conditions that result in an increase in total levels of β-catenin in β-cells are known to induce cell proliferation [33,48,49]. In β-cells this would probably be a consequence of the increased expression of cyclin D1 [20,50]. There is evidence that β-catenin phosphorylated at Ser552 by PKA is also capable of inducing proliferation in other cell types . In addition, the elevated levels of β-catenin are also likely to regulate FOXO (forkhead box O) signalling . FOXOs play an important role in regulating β-cell function , and it has been shown in other tissues that β-catenin interacts with FOXO and regulates expression of metabolic genes (e.g. in liver ). What we observed in the present study is that inhibiting β-catenin supresses GSIS. Some previous studies also found that the Wnt pathway was involved in insulin secretion [15,17,18]. The increase in β-catenin can also play roles in regulating insulin secretion, as TCF7L2 is required for both GSIS and the ability of cAMP-inducing agents to potentiate insulin secretion . Fujino et al.  also showed that Wnt/LRP5 [LDL (low-density lipoprotein) receptor-related protein 5] signalling contributes to GSIS in the islets. They found that LRP5-knockout mice exhibit an impaired GSIS, and that Wnt3a and Wnt5a improve this. Furthermore, mice with deletion of β-catenin, specifically in β-cells, have a defect in GSIS , as islets from these mice secrete less insulin in response to glucose and surviving transgenic adults exhibit a defective GSIS. The results of the present study showing that inhibition of β-catenin in β-cells disrupts GSIS supports the findings of these previous studies [17,18,55], and extends them by identifying a mechanism by which glucose actually regulates β-catenin through elevation of cAMP levels.
Analysis of previous literature reveals two possible mechanisms by which the change in β-catenin levels or the change in β-catenin phosphorylation status might regulate insulin secretion. The first is that this may play a role in regulating the localization of insulin-containing vesicles, since a role for β-catenin in synaptic vesicle localization has been described [56,57]. In support of this, β-catenin is found to co-localize with insulin granules at cell junctions in MIN6 pseudoislets . Another explanation would be a regulation of ion fluxes, since there is a significant body of evidence that β-catenin can regulate the location and/or function of ion transporters at the plasma membrane in β-cells [59–63]. The resulting changes in ion currents could affect insulin granule release. One candidate might be Kir6.2, the ATP-sensitive potassium channel known to be involved in GSIS, but there are no reports of Kir6.2 associating with β-catenin. Another candidate would be TRP (transient receptor potential) channels which have also been implicated in regulating insulin secretion; some members of this family are known to associate with β-catenin . Either a β-catenin-dependent effect on vesicle location or an effect on ion channels could provide plausible mechanisms linking intracellular glucose levels and insulin release mechanisms. This would also provide a unified explanation as to how agents which increase cellular cAMP levels, such as GLP-1, can potentiate insulin secretion (Figure 9). Another interesting feature of this model is that it could potentially explain the observation that overexpression of TCF7L2 attenuates a late stage in insulin secretion [16,18], as the binding of TCF-7L2 to β-catenin would block it binding to other partners and would divert β-catenin from the cell membrane to the nucleus.
In summary, the present study shows that β-catenin signalling is regulated by changes in glucose levels in β-cell models and shows that activation of the cAMP/PKA pathway is required for this to occur. Taken together, the results of the present study provide evidence that β-catenin can act as a mediator of nutrient sensing in β-cells.
Emmanuelle Cognard and Peter Shepherd contributed to the experimental design. Emmanuelle Cognard, Coralie Dargaville and Deborah Hay performed the experiments. Deborah Hay contributed analytic tools. Emmanuelle Cognard, Peter Shepherd and Deborah Hay analysed and interpreted the data. Emmanuelle Cognard and Peter Shepherd wrote the paper.
This work was supported by the Health Research Council of New Zealand [grant number 08/076].
Abbreviations: AICAR, 5-amino-4-imidazolecarboxamide riboside; AKAPs, A-kinase-anchoring protein; AMPK, AMP-activated protein kinase; APC, adenomatous polyposis coli; CK1, casein kinase 1; CREB, cAMP-response-element-binding protein; db-AMP, dibutyryl-cAMP; ddAdo, 2′,3′-dideoxyadenosine; FBS, fetal bovine serum; FOXO, forkhead box O; GLP-1, glucagon-like peptide-1; GSIS, glucose-stimulated insulin secretion; GSK3, glycogen synthase kinase 3; IBMX, isobutylmethylxanthine; LRP5, LDL (low-density lipoprotein) receptor-related protein 5; PKA, cAMP-dependent protein kinase/protein kinase A; PKA-Cα, the catalytic subunit α of PKA; qRT-PCR, quantitative real-time-PCR; siRNA, small interering RNA; TCF7L2, transcription factor 7-like 2
- © The Authors Journal compilation © 2013 Biochemical Society