Glucose-Induced Nuclear Shuttling of ChREBP Is Mediated by Sorcin and Ca2+ Ions in Pancreatic β-Cells
- Section of Cell Biology, Division of Diabetes, Endocrinology and Metabolism, Department of Medicine, Faculty of Medicine, Imperial College London, London, U.K
- Corresponding authors: Guy A. Rutter, email@example.com, and Isabelle Leclerc, .
N.A.N. and G.M. contributed equally to this study.
Carbohydrate-responsive element-binding protein (ChREBP) is a regulator of pancreatic β-cell gene expression and an important mediator of glucotoxicity. Glucose increases the activity and nuclear localization of ChREBP by still ill-defined mechanisms. Here we reveal, using both MIN6 and primary mouse β-cells, a unique mechanism behind ChREBP nuclear translocation. At low glucose concentrations, ChREBP interacts with sorcin, a penta EF hand Ca2+ binding protein, and is sequestered in the cytosol. Sorcin overexpression inhibits ChREBP nuclear accumulation at high glucose and reduced the activity of L-type pyruvate kinase (L-PK) and TxNIP promoters, two well-characterized ChREBP target genes. Sorcin inactivation by RNA interference increases ChREBP nuclear localization and in vivo binding to the L-PK promoter at low glucose concentrations. Ca2+ influx was essential for this process since Ca2+ chelation with EGTA, or pharmacological inhibition with diazoxide and nifedipine, blocked the effects of glucose. Conversely, mobilization of intracellular Ca2+ with ATP caused the nuclear accumulation of ChREBP. Finally, sorcin silencing inhibited ATP-induced increases in intracellular Ca2+ and glucose-stimulated insulin secretion. We therefore conclude that sorcin retains ChREBP in the cytosol at low glucose concentrations and may act as a Ca2+ sensor for glucose-induced nuclear translocation and the activation of ChREBP-dependent genes.
Carbohydrate-responsive element-binding protein (ChREBP) is a member of the basic helix-loop-helix family of transcription factors and transactivates glucose-responsive genes by binding to DNA as a heterodimer with Max-like protein ×1 at a well-defined carbohydrate-responsive element (1,2). ChREBP is responsible for converting excess carbohydrate to fatty acids for long-term storage (1). Thus, ChREBP-null mice display diminished rates of hepatic glycolysis and lipogenesis resulting in high liver glycogen content, low plasma free fatty acid levels, and reduced adipose tissue mass (3). Loss of ChREBP in ob/ob mice protects against obesity and hyperphagia (3,4). In the pancreatic β-cell, ChREBP overexpression (5) or silencing (6) results in the inhibition and stimulation, respectively, of glucose-stimulated insulin secretion (GSIS). These actions of ChREBP seem likely to be mediated through alterations in the deleterious effects of high glucose concentrations on β-cell function and survival, termed glucotoxicity. The latter process may involve the induction by ChREBP of lipogenic genes such as fatty acid synthase and L-type pyruvate kinase (L-PK) (6,7), as well as the proapoptotic gene thioredoxin interacting protein Txnip (8,9), and suppression of critical β-cell genes such as aryl hydrocarbon receptor nuclear translocator/hypoxia inducible factor-1β (ARNT/HIF-1β) (5), Pdx-1, MafA, glucokinase, and insulin (10).
Translocation of ChREBP from the cytosol to the nucleus in response to elevated glucose concentrations has been documented in both liver (11) and clonal pancreatic β-cells (6,12,13) and is likely to be a key regulatory step in the activation of downstream genes. Although previous work in the pancreatic β-cell line INS1(832/13) has identified a glucose-sensing module in the NH2-terminal region (12,13), the exact mechanism(s) through which glucose prompts this subcellular relocalization are still unknown (14).
Sorcin (soluble resistance-related calcium binding protein) is a 22 kDa protein, highly conserved among mammals and highly expressed in primary mouse islets (top 10th percentile of all messages as assessed by microarray and RNAseq data, G. Rutter, unpublished observations) that belongs to the penta-EF-hand family (15,16). In common with other members of this family, sorcin undergoes Ca2+-dependent conformational changes and translocation from cytosol to membranes where it binds to target proteins (17,18). In cardiac and skeletal muscle, sorcin interacts with the ryanodine receptor (RyR) (19) and with the pore-forming α1-subunit of voltage-dependent L-type Ca2+ channels (20). Sorcin has been shown to inhibit RyR activity (21), and therefore to play a role in terminating the Ca2+-induced Ca2+ release process (22,23), which would otherwise feed forward and deplete intracellular Ca2+ stores (24). It is noteworthy that sorcin phosphorylation by cAMP-dependent protein kinase (PKA) significantly decreases its ability to modulate RyR activity (21).
Here we show that, in the excitable pancreatic β-cell, ChREBP is sequestered in the cytosol through sorcin binding at low glucose concentrations. Upon stimulation with glucose and activation of Ca2+ influx, or application of ATP as an intracellular Ca2+-mobilizing agent, ChREBP rapidly translocates to the nucleus. In sorcin-silenced cells, ChREBP is constitutively present in the nucleus, and both glucose and Ca2+ are ineffective in stimulating further ChREBP nuclear shuttling.
RESEARCH DESIGN AND METHODS
Plasmids and viruses.
Plasmids c-myc–ChREBP(WT) and c-myc–Pdx-1 have been described elsewhere (5,25). Plasmid c-myc–ChREBP(1–242) was generated by digesting c-myc–ChREBP(WT) by XbaI and self-ligation. Plasmids pGBKT7-ChREBP and pChREBP-enhanced green fluorescent protein (EGFP) were generated by subcloning full-length ChREBP cDNA in frame with the Gal4 DNA binding domain and EGFP, respectively, at the Sal1 sites of pGBKT7 and pEGFP-C2 (Clontech). Plasmid sorcin-HA was derived from the yeast vector pGADT7 (see below) using the following primers: forward, 5′-CGA AGC TTA TGG CGT ATC CCG GGC AC-3′ and reverse, 5′-ATG TGC GGC CGC GGC ATA GTC TGG GAC GTC ATA TGG ATA GAC GGT CAT GAC ACA CTG GAT G-3, HA tag underlined, and subcloned at SalI and NotI sites of pcDNA3.1+ (Invitrogen). Plasmids −183LPK.luci and −148LPK.luci have been described in Ref. 26. Plasmid −1081TXNIP.luci was obtained from Addgene and described in Ref. 27. Adenovirus sorcin-HA, also expressing GFP from a single bicistronic mRNA under the cytomegalovirus (CMV) promoter, was generated by three-piece ligation of sorcin-HA and IRES-EGFP in pShuttle-CMV (28) using BglII, XhoI, and XbaI restriction sites. Adenovirus production was then performed as in Ref. 28. Sorcin (sc-41017-v) and control (sc-108080) short hairpin RNA (shRNA) mouse lentiviruses were purchased from Santa Cruz. All constructs were verified by DNA sequencing.
Cell culture and transfection.
Human embryonic kidney 293, MIN6, and INS1 (832/13) cells were cultured as in Ref. 5 unless specified otherwise. Cells were transfected using Lipofectamine 2000 for plasmids and RNAiMAX for small interfering RNA (siRNA) (final concentration 2.5 to 5 nM) in Opti-Mem (Invitrogen).
MIN6 β-cells cDNA library generation.
MIN6 cDNA library was generated according to Matchmaker Library Construction and Screening Kit (Clontech) using Oligo d(T) primers, 2.0 μg total RNA, yeast strain AH109, and pGADT7-Rec AD. The transformants were selected on SD/-leu plates before yeast mating.
Yeast two-hybrid was performed according to Matchmaker Gal4 Two-Hybrid System 3 (Clontech). In brief, yeast mating was performed using yeast strain AH109 containing the MIN6 cDNA library, and yeast strain Y187 was pretransformed with pGBKT7-ChREBP. Screens were conducted at high (-adenine/-histidine/-leucine/-tryptophan) stringency with 5 mmol/L 3-AT to prevent background growth. The library cDNAs contained in pGADT7 were identified by sequencing.
Coimmunoprecipitation and Western (immuno-) blotting.
Cells were transfected with plasmid vectors encoding c-myc–ChREBP(WT), c-myc–ChREBP(1–242), c-myc–Pdx-1 and sorcin-HA, or sorcin-HA alone for 48 h before coimmunoprecipitation with mouse anti–c-myc and Western blotting as described in Ref. 29 using either mouse anti–c-myc, mouse anti-HA, or mouse anti-sorcin antibodies.
Chemical crosslinking analysis.
MIN6 β-cells were cultured for 16 h in 3 mmol/L glucose followed by 3 h in 3 or 30 mmol/L glucose. The cells were washed twice with PBS and incubated with 1 mmol/L disuccinimidyl suberate (Pierce), at room temperature, for 30 min. The crosslinker reaction was quenched by incubating with 20 mmol/L Tris (pH 7.4) for 15 min at room temperature, followed by washing twice with ice-cold PBS. The cells were lysed in Nonidet-40 lysis buffer before immunoprecipitation with anti-sorcin antibody and Western blotting as above.
Chromatin immunoprecipitation assay.
MIN6 β-cells were transfected with scrambled or specific sorcin siRNA for 48 h. Chromatin immunoprecipitation was performed using anti-ChREBP antibody and mouse L-PK promoter primers as described (5,6).
Nuclear-cytosolic protein extraction.
MIN6 β-cells, nontransfected or transfected with sorcin-HA for 48 h, were cultured for 16 h in 3 mmol/L glucose followed by 6 h in 3 or 30 mmol/L glucose. The cells were scraped in ice-cold PBS, followed by centrifuging at 500g for 2 min. The cell pellets were lysed, and nuclear proteins were separated from the cytosolic fraction using the NucBuster Protein Extraction Kit (Novagen) before Western blotting.
MIN6 β-cells seeded in 24-well plates were transfected with 1.5 μg of either −183LPK.luci, −148LPK.luci, or −1081TXNIP.luci reporter plasmids, 0.5 μg of either GFP or sorcin cDNA, and 1.5 ng of renilla luciferase to correct for transfection efficiency, using Lipofectamine 2000 (Invitrogen) and OPTI-MEM (5 mmol/L glucose) for 24 h followed by 25 mmol/L glucose Dulbecco’s modified Eagle’s medium for 24 h before cell lysis and luciferase assay using Dual-Luciferase Reporter Assay System (Promega).
Immunostaining and confocal microscopy.
MIN6 β-cells were fixed in 4% (volume for volume) paraformaldehyde, permeabilized with 0.3% Triton X-100, and stained with rabbit anti–c-myc antibody (1:250; Santa Cruz), and mouse anti-HA antibody (1:1000; 12CA5, Roche), followed by Alexa 488 conjugated goat anti-rabbit IgG and Alexa 568 conjugated donkey anti-mouse IgG (1:1,000; Invitrogen). Cells were imaged using a Zeiss Axoivert 200M microscope fitted with a PlanApo ×63 oil immersion objective. Confocality was achieved using a Nipkow spinning disc under the control of Volocity 4.0 (Improvision) software.
Measurement of free cystolic intracellular Ca2+.
MIN6 β-cells were loaded with fura-2 a.m. (2 μmol/L in DMSO) in Krebs-Ringer bicarbonate HEPES (KRBH) buffer (125 mmol/L NaCl, 3.5 mmol/L KCl, 1.5 mmol/L CaCl2, 0.5 mmol/L MgSO4, 0.5 mmol/L NaH2PO4, 25 mmol/L NaHCO3, and 10 mmol/L HEPES), supplemented with 3 mmol/L glucose for 60 min at 37°C. Cells were perifused in KRBH and imaged at 37°C using an Olympus IX-70 with an Imago charge-coupled device camera (Till Photonics, Grafelfing, Germany) controlled by TILLvisION software (Till Photonics). Cells were illuminated alternately for 5 ms at 340 and 380 nm, and the emission was filtered at 510 nm. After background and auto-fluorescence were subtracted (by quenching with 10 mmol/L MnCl2 and 1 μmol/L ionomycin), free cystolic intracellular Ca2+ ([Ca2+]cyt) was calculated using a look-up table created from standard Ca2+ solutions (Molecular Probes).
Live-cell confocal imaging of ChREBP-EGFP.
MIN6 β-cells were transfected either with ChREBP-EGFP plasmid alone or with sorcin siRNA for 48 h. Cells were then incubated overnight with 3 mmol/L glucose Dulbecco’s modified Eagle’s medium, and subsequently the coverslips were rinsed with 3 mmol/L glucose KRBH buffer and mounted on a hot stage with continuous perifusion with warm KRBH. Cells were imaged using a Zeiss Axiovert 200M microscope (Carl Zeiss, Jena, Germany) fitted with a PlanApo 63× oil-immersion objective and a 1.5 Optivar attached to a Nokigawa spinning disc confocal head. Hoechst (Invitrogen) staining of nuclei was visualized through DAPI filter. GFP-expressing samples were illuminated using steady-state 488 nm laser line, and emission was collected through ET535/30 emission filter (Chroma) for every 5 s. Images were captured using a Hamamatsu EM charge-coupled device digital camera, model C9100-13, controlled by Volocity 4.0 software. Deconvoluted images were analyzed by obtaining the gray values for every time point in the region of interest drawn around nucleus and the cytosol, which was then corrected for background and normalized, and ratios of nuclear to cytosolic distribution were generated.
Preparation and imaging of dissociated mouse islets.
Mouse islets were isolated (5) and partially dissociated by incubation in Hank’s based cell dissociation buffer (Invitrogen) for 10 min by gentle pipetting. To assess the effects of intracellular Ca2+, primary cell clusters were transduced with c-myc–ChREBP-expressing adenoviral particles (also expressing GFP) (5) at a multiplicity of infection of ∼50 for 6 h, followed by 48 h culture in fresh medium. Dissociated islets were then incubated overnight in 3 mmol/L glucose and thereafter in 3 or 17 mmol/L glucose in KRBH buffer containing either 1.5 mmol/L CaCl2 or 10 mmol/L EGTA for 30 min and fixed in 4% paraformaldehyde for 10 min. After permeabilization with 0.1% Triton × 100, immunostaining was carried out using the following antibodies: monoclonal anti–c-myc antibody (1:200; Roche), polyclonal anti-swine insulin (1:200; DakoCytomation, Ely, UK), and Alexa 564 or 633-coupled secondary antibodies (1:1,000; Molecular Probes), and coverslips were mounted in VECTASHIELD (Vector Laboratories) hardset medium with DAPI. Imaging was performed using a Leica SP2 upright confocal microscope with an HCX PL APO 63× 1.32 numerical aperture oil immersion objective and the following laser lines: Argon ion (488 nm), HeNe (633 nm), 561 nm diode, and ultraviolet (350 nm) controlled by Leica LAS AF Lite software. Acquired images were analyzed using Volocity software (Improvision). To assess the effect of sorcin overexpression or silencing on endogenous ChREBP nuclear localization, dissociated islets were transduced with either sorcin (also expressing GFP) or GFP adenoviral particles as above or with shRNA sorcin or control lentiviral particles (Santa Cruz) according to the manufacturer’s instructions in 11 mmol/L glucose RPMI 1640 for 48 h followed by 1 h in 3 mmol/L glucose RPMI 1640 and 6 h in 17 mmol/L glucose RPMI 1640. Cells were then fixed and immunostained using rabbit anti-ChREBP (1:100; Thermo Scientific) and mouse anti-sorcin (1:50; Invitrogen) and secondary goat–anti-rabbit Alexa 568, and goat–anti-mouse Alexa 633 (both at 1:1,000; Invitrogen).
Insulin secretion and assay.
MIN6 β-cells were transfected with sorcin-specific or scrambled siRNAs for 48 h before insulin secretion assay (5).
Data are given as mean ± SEM of at least three individual experiments. Comparisons between means were performed using two-tailed Student t test for unpaired data, with Bonferroni correction and ANOVA.
ChREBP and sorcin are molecular binding partners under physiological conditions.
We first detected sorcin as a ChREBP interactor in a yeast two-hybrid screen using full length ChREBP as bait in a pancreatic β-cell MIN6 library (not shown). We confirmed the interaction in mammalian cells by coimmunoprecipitation experiments of overexpressed c-myc–tagged ChREBP and HA-tagged sorcin in human embryonic kidney 293 cells (Supplementary Fig. 1) and INS-1(832/13) β-cells (Fig. 1A, first lane from left). This interaction was further delimited to the first 242 amino acids of ChREBP (Fig. 1A, second lane), a region known as the low-glucose inhibitory domain (12). Establishing the specificity of the ChREBP-sorcin interaction, coimmunoprecipitation experiments did not reveal any interaction between sorcin-HA and c-myc–Pdx-1 (Fig. 1A, fourth lane), a transcription factor that also translocates to the nucleus in response to high glucose in this cell type (25).
We next explored the interaction between endogenous ChREBP and sorcin in MIN6 cells. Such interaction was only detectable after crosslinking experiments, as shown in Fig. 1B. Indeed, crosslinking and immunoprecipitation with anti-sorcin antibody revealed a high molecular complex of ∼140 kDa using both anti-sorcin (Fig. 1B, top panel, second and third lanes) and anti-ChREBP (Fig. 1B, bottom panel, second and third lanes) antibodies. We believe this complex to represent sorcin dimers and ChREBP, since sorcin dimers were also detected after addition of the crosslinker (Fig. 1B, middle panel, second and third lanes).
Sorcin acts as a cytosolic anchor for ChREBP.
It has been shown previously that ChREBP constantly shuttles between the cytosol and the nucleus (13). The cytosolic retention domain has been mapped to the NH2-terminal region of ChREBP, since NH2-terminal deletion mutants lacking the first 252 amino acids are predominantly nuclear (12,30,31).
Because sorcin interacts with ChREBP specifically through this domain (see Fig. 1A), we next investigated whether sorcin may play a role in controlling the subcellular localization of ChREBP. With the use of spinning disc confocal microscopy, c-myc–ChREBP immunoreactivity was examined in MIN6 β-cells transfected with either c-myc–ChREBP alone or cotransfected with c-myc–ChREBP and sorcin-HA and incubated at low or high glucose concentrations. As expected from the coimmunoprecipitation experiments presented above, ChREBP and sorcin immunoreactivities extensively colocalized, especially at low glucose concentrations (Fig. 2B). Strikingly, however, sorcin overexpression greatly diminished the abundance of ChREBP staining in the nucleus from 25.7 ± 2.1% in the control cells to 9.4 ± 0.8% in the sorcin overexpressed cells (P < 0.005, Fig. 2C), suggesting that sorcin may serve as a cytosolic anchor for ChREBP. This was also observed in cell fractionation experiments where sorcin overexpression reduced ChREBP immunoreactivity in the nuclear compartment of cells incubated in elevated glucose concentration (Fig. 2D). Moreover, sorcin overexpression significantly reduced the activity of the −183L-PK and TXNIP promoters, two well-characterized ChREBP target genes (6,9) in luciferase reporter assays, whereas a truncated L-PK promoter (−148L-PK), lacking the ChREBP binding site, was unaffected by sorcin (Fig. 2E).
We next examined the effects of sorcin silencing on ChREBP binding to the L-PK promoter by chromatin immunoprecipitation (ChIP) and subcellular localization in response to glucose. Using siRNA, we achieved a reduction of ∼86% of sorcin immunoreactivity (Fig. 3A and B). As reported previously (6), glucose (30 vs. 3 mmol/L) stimulation increased ChREBP binding on the proximal L-PK promoter (Fig. 3C, top panel). In contrast, in sorcin-silenced cells, ChREBP binding to the L-PK promoter was constitutive, irrespective of glucose concentrations (Fig. 3C, bottom panel, and Fig. 3D for quantification). Accordingly, inactivation of sorcin by RNAi led to an increase in the number of cells displaying nuclear ChREBP-EGFP fluorescence at resting (3 mmol/L) glucose concentration (83.3 ± 2.9% vs. 23.3 ± 4.0%, P < 0.001; Fig. 3E and F).
These results designate sorcin as a physiological regulator of ChREBP activity in response to glucose in MIN6 cells.
Elevated glucose concentrations, Ca2+ influx, and sorcin are all necessary for ChREBP nuclear translocation.
Because sorcin undergoes conformational changes after Ca2+ binding (18), we next tested whether Ca2+ influx in response to glucose or other agents would drive ChREBP into the nucleus. Imaged dynamically in living MIN6 β-cells, elevated glucose concentrations caused a rapid translocation of ChREBP-EGFP from the cytosol to the nucleus (Fig. 4A and G and Supplementary Movie 1). Parallel single-cell measurements of [Ca2+]cyt revealed that nuclear accumulation of ChREBP closely followed (∼1 min) a glucose-evoked Ca2+ signal (Δ[Ca2+]cyt = 312 ± 14 nmol/L; Fig. 3G). Chelation of extracellular Ca2+ with 1 mmol/L EGTA (Fig. 4B and G and Supplementary Movie 2) abolished glucose-induced Ca2+ entry and thereby Ca2+-induced nuclear translocation of ChREBP, which was also suppressed by 10 μmol/L nifedipine, an L-type Ca2+ channel blocker, and by 250 μmol/L diazoxide, an activator of ATP-sensitive K+ channels (Fig. 4C and G and Supplementary Movie 3) that prevent membrane depolarization and subsequent Ca2+ entry. Furthermore, a transient Ca2+ peak (Δ[Ca2+]cyt = 108 ± 11 nmol/L) elicited by 250 μmol/L extracellular ATP, an activator of G protein–coupled P2 purinergic receptors (32), led to robust cytosol-nuclear shuttling of ChREBP in the absence of extracellular Ca2+ (Fig. 4D and H and Supplementary Movie 4), suggesting that mobilization of intracellular Ca2+ stores was sufficient for translocation. Apparently, nuclear translocation of ChREBP also depended upon the presence of elevated concentrations (30 mmol/L) of glucose (Fig. 4E and Supplementary Movie 5), but this largely reflected the increased Ca2+ release (Δ[Ca2+]cyt = 31 ± 7 nmol/L at low vs. 108 ± 11 nmol/L at high glucose) in these conditions (Fig. 4H).
We next examined the effects of sorcin silencing on ChREBP nuclear translocation in response to elevated glucose concentrations and Ca2+ signals. Although glucose-induced Δ[Ca2+]cyt were not significantly different (330 ± 20 vs. 280 ± 22 nmol/L for control and sorcin-silenced cells, P = 0.55), glucose stimulation failed to trigger nuclear shuttling of ChREBP in sorcin-silenced cells (Fig. 4F and Supplementary Movie 6). On the contrary, a high level of ChREBP was already detected in the nucleus, even before treatments, pointing to the role of sorcin as a ChREBP cytosolic anchor as discussed above. Silencing of sorcin also completely abolished ATP-evoked Ca2+ release from intracellular stores (Δ[Ca2+]cyt = 234 ± 20 nmol/L for control vs. −54 ± 18 nmol/L for sorcin-siRNA; Fig. 4I).
Sorcin knockdown inhibits GSIS.
We next examined the impact of sorcin inhibition on GSIS. As shown in Fig. 5, sorcin silencing significantly decreased GSIS in pancreatic MIN6 β-cells.
Calcium influx is required to cause ChREBP nuclear translocation in primary mouse β-cells in response to glucose.
We next examined the effects of Ca2+ influx and sorcin in primary β-cells. Dissociated CD1 mouse islets were transduced with c-myc–tagged ChREBP-expressing adenoviral particles (5) for 48 h and incubated overnight in 3 mmol/L glucose and thereafter in 3 or 17 mmol/L glucose KRBH containing either 1.5 mmol/L CaCl2 or 10 mmol/L EGTA for 30 min before immunostaining and imaging as described in research design and methods. The identity of the β-cells was established by simultaneous immunostaining with insulin antibody. As shown in Fig. 6, inhibiting Ca2+ influx also abolished ChREBP nuclear localization in response to glucose.
Sorcin controls ChREBP subcellular location in primary mouse β-cells.
Extending the physiological relevance of the above findings, sorcin overexpression in dissociated primary mouse islets (comprising 70–80% β-cells) (33) diminished endogenous ChREBP nuclear immunoreactivity from 28.6 ± 1.5% in control cells to 16.5 ± 1.0% in sorcin overexpressing cells incubated in elevated (17 mmol/L) glucose (P < 0.001, Fig. 7A and B). Conversely, sorcin silencing increased endogenous ChREBP nuclear staining to 25.5 ± 1.4% compared with 13.6 ± 1.1% in control cells (P < 0.001, Fig. 7C and D), incubated in low (3 mmol/L) glucose.
The chief goal of the current study was to elucidate the molecular mechanisms through which elevated glucose concentrations lead to the nuclear accumulation of ChREBP in the pancreatic β-cell. We show that sorcin provides a Ca2+-regulated cytosolic anchor, likely to play an important role in the regulation of this process in both clonal and primary β-cells.
In the liver, glucose-dependent ChREBP nuclear translocation is subject to a series of phosphorylation/dephosphorylation reactions. In fasting and in low glucose conditions, ChREBP is phosphorylated by PKA on S196 and T666 and by AMP-activated protein kinase on S568, which promote cytosol retention and inhibit DNA binding. By contrast, in high glucose conditions, protein phosphatase 2A activation by xylulose 5-phosphate (34) induces dephosphorylation of S196, T666, and S568; nuclear import; and DNA binding (35,36). Dephosphorylation of these residues, however, was not sufficient for ChREBP nuclear translocation in the glucose-responsive pancreatic β-cell line INS1 (832/13) (13). This is unsurprising, since, in the pancreatic β-cell, elevated glucose concentrations are rather associated with protein phosphatase 2A inhibition (37,38) and increased intracellular cAMP levels and PKA signaling (39,40), pointing to the existence of additional mechanisms, possibly including the inhibition by glucose of AMP-activated protein kinase (41).
Here we show that, in the excitable pancreatic β-cell, elevated glucose concentrations provoke ChREBP nuclear translocation through stimulation of Ca2+ influx. Thus, extracellular Ca2+ chelation with EGTA or pharmacological blockade of Ca2+ entry with diazoxide or nifedipine completely abolished glucose-induced ChREBP nuclear shuttling, both in clonal MIN6 (Fig. 4) and primary mouse β-cells (Fig. 6). Conversely, mobilization of intracellular Ca2+ with extracellular ATP was sufficient to cause ChREBP nuclear translocation. Together, these data point to a central role for [Ca2+]cyt in this phenomenon. Critically, we show that overexpression or silencing of sorcin in clonal (Fig. 2–4) or primary (Fig. 7) β-cells blocks and inhibits, respectively, nuclear accumulation of ChREBP, implicating sorcin as a likely sensor of cytosolic Ca2+.
Changes in [Ca2+]cyt have been reported previously to be detected and decoded as nuclear events via mechanisms involving cytosol-nuclear translocation of nuclear factor activator of transcription and nuclear factor-κB cells (NF-κB) (42). The signaling module reported here may represent a similar system, which allows highly localized changes in cytosolic Ca2+, perhaps close to the ER or even a specific Ca2+ release channel, to be decoded as a transcriptional response. We thus propose a model (Fig. 8) in which sorcin, a penta-EF-hand protein that undergoes Ca2+-dependent conformational changes (15–18), binds to and sequesters ChREBP in the cytosol at low glucose, through an interaction with the NH2-terminal domain of ChREBP. After glucose stimulation and Ca2+-induced Ca2+ release, sorcin liberates ChREBP, which is then free to translocate to the nucleus. The regulation of ARNT/HIF-1β gene in pancreatic β-cells supports this model of ChREBP activation. Indeed, we have recently shown that ChREBP is a repressor of ARNT/HIF-1β promoter in pancreatic β-cells (5), whereas another group (43) has shown that blocking Ca2+ flux from intracellular Ca2+ channels induces the expression of ARNT/HIF-1β in this cell type.
Other interesting findings of this study are the apparent depletion of intracellular Ca2+ stores (Fig. 4I) and the inhibition of GSIS (Fig. 5) after sorcin silencing in MIN6 cells. The absence of sorcin may lead to a depletion of intracellular Ca2+ stores by allowing random activation of RyR (21). Another possibility would be a change in gene expression, possibly the expression of the G protein–coupled P2 purinergic receptors, hence diminishing the response to extracellular ATP. However, any such changes were apparently efficiently countered by modifications to Ca2+ pumping since neither resting nor glucose-stimulated increases in cytosolic Ca2+ were perturbed by the knockdown of sorcin (Fig. 4I). It follows that the interaction of sorcin with voltage-dependent (L-type) Ca2+ channels (20) in the β-cell may be of limited importance in the control of Ca2+ influx. The latter observation argues against substantial changes in β-cell glucose metabolism or in cellular viability after sorcin silencing in the short period (48 h) of our study. However, a chronic sorcin inhibition is likely to induce changes in gene expression through a constitutive ChREBP signaling pathway, including downregulation of ARNT/HIF-1β (5) and upregulation of TxNIP (9). Although the latter is often associated with oxidative stress and cell death by mechanisms involving mitochondrial Ca2+ accumulation (44), we would note that the possible depletion of ER Ca2+ stores (see above), required in several cell types for efficient accumulation of mitochondrial Ca2+ (45), may reduce the magnitude of any proapoptotic effects of TxNIP induction. Further detailed studies will be required in the future to discriminate between these possibilities.
An event distal to increase in [Ca2+]cyt is therefore likely to be involved in the inhibition of GSIS. This may, for example, include the less efficient mobilization or fusion of secretory granules with the plasma membrane in sorcin-silenced cells (46).
An exciting corollary of the work presented here is the development of novel therapeutic approaches involving sorcin activators or sorcin overexpression in the treatment of failing β-cells in type 2 diabetes, which may complement the positive action of such agents in the heart (47,48).
This work was supported by grants from Diabetes UK (BDA:RD04/0002895) and the Wellcome Trust (WT082366MA) to I.L. and by the Wellcome Trust (Programme Grant 081958/2/07/Z), the European Community's Seventh Framework Programme (FP7/2007-2013) for the Innovative Medicine Initiative under grant agreement n° 11500 (IMIDIA) to G.A.R.
No potential conflicts of interest relevant to this article were reported.
N.A.N., G.M., and I.L. designed the experiments, researched data, wrote the manuscript, and contributed to the discussion. G.A.R. designed the experiments, reviewed and edited the manuscript, and contributed to the discussion. As the corresponding author and guarantor of this article, I.L. takes full responsibility for the work as a whole, including the study design, access to data, and the decision to submit and publish the manuscript.
This study was presented as an oral communication at the 46th Annual Meeting of the European Association for the Study of Diabetes, Stockholm, Sweden, 20–24 September 2010; the Congrès de la Société Francophone de Diabète, Geneva, Switzerland, 22–25 March 2011; the Annual Professional Conference of Diabetes UK, London, U.K., 30 March–1 April 2011; and the 28e Congrès de la Société Française d’Endocrinologie, Clermont-Ferrand, France, 10–13 October 2011.
The authors thank Prof. Jean-Claude Henquin, University of Louvain Faculty of Medicine, Brussels, Belgium, for helpful discussion.
This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1329/-/DC1.
- Received September 17, 2010.
- Accepted November 21, 2011.
- © 2012 by the American Diabetes Association.
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