Translationally controlled tumour protein (TCTP) is a novel glucose-regulated protein that is important for survival of pancreatic beta cells
This study used proteomics and biochemical approaches to identify novel glucose-regulated proteins and to unveil their role in pancreatic beta cell function. Translationally controlled tumour protein (TCTP) was identified to be one such protein, and further investigations into its function and regulation were carried out.
Global protein profiling of beta cell homogenates following glucose stimulation was performed using two-dimensional gel electrophoresis. Proteins were identified by mass spectroscopy analysis. Immunoblotting was used to investigate alterations in TCTP protein levels in response to glucose stimulation or cell stress induced by palmitate. To investigate the biological function of TCTP, immunolocalisation, gene knockdown and overexpression of Tctp (also known as Tpt1) were performed. Apoptosis was measured in Tctp knockdown or Tctp-overexpressing cells. Glucose-stimulated insulin secretion was carried out in Tctp knockdown cells.
TCTP was identified as a novel glucose-regulated protein, the level of which is increased at stimulatory glucose concentration. Glucose also induced TCTP dephosphorylation and its partial translocation to the mitochondria and the nucleus. TCTP protein levels were downregulated in response to cell stress induced by palmitate or thapsigargin treatments. Gene knockdown by small interfering RNA led to increased apoptosis, whereas overproduction of TCTP prevented palmitate-induced cell death.
Regulation of TCTP protein levels by glucose is likely to be an important cyto-protective mechanism for pancreatic beta cells against damage caused by hyperglycaemia. In contrast, high concentration of palmitate causes cell stress, reduction in TCTP levels and consequently reduced cell viability. Our results imply that TCTP levels influence the sensitivity of beta cells to apoptosis.
KeywordsApoptosis Fatty acid palmitate Insulin secretion Proteomics analysis TCTP Translationally controlled tumour protein
BCL2-associated X protein
B cell lymphoma 2
Two-dimensional gel electrophoresis
Extracellular signal regulated kinase
Glyceraldehyde 3-phosphate dehydrogenase
Immobilised pH gradient
Myeloid cell leukaemia sequence 1
PKR-like ER kinase
Double-stranded RNA-dependent protein kinase
Small interfering RNA
Translationally controlled tumour protein
Unfolded protein response
Using a proteomics-based approach, we identified and characterised glucose-regulated proteins in pancreatic beta cells. Global analysis of protein levels using proteomics can unveil biologically important changes in protein production, which may not be identified by cDNA microarray analysis. The latter method is limited by the fact that mRNA levels and not their protein products are investigated. Gene expression studies based on cDNA microarrays do not always correlate with changes found at the protein level [1, 2, 3]. Furthermore, many proteins are also regulated at post-transcriptional levels, effecting changes that transcriptome analysis would fail to identify. Our proteomics-based analysis identified translationally controlled tumour protein (TCTP) as a glucose-regulated protein in pancreatic beta cells.
TCTP is a highly conserved protein of 23 kDa [4, 5], which shows no sequence similarity with any other known proteins. TCTP has been identified in a wide range of eukaryotic organisms and has been associated with diverse cellular processes, as reviewed by others . However, the physiological function of TCTP in humans is still not fully elucidated. TCTP has been suggested to function as an anti-apoptotic protein, since overproduction of the protein inhibits, whereas knockdown of its gene promotes apoptosis [7, 8, 9, 10, 11, 12]. Gene knockout studies revealed that TCTP-deficient mice [10, 13] and TCTP-deficient mutants of Drosophila  die early during embryogenesis, presumably due to unregulated apoptosis at a critical stage. It has been shown that TCTP binds to the anti-apoptotic members of the B cell lymphoma 2 (BCL2) family of proteins, myeloid cell leukaemia sequence 1 (MCL-1) [7, 8] and B cell lymphoma extra large (BCL-XL) . It has recently been proposed that TCTP antagonises apoptosis by enhancing the anti-apoptotic actions of MCL-1 and BCL-XL, and by anchoring into the mitochondrial membrane in a way that inhibits dimerisation of the proapoptotic protein BCL2-associated X protein (BAX) . Thus, the above-mentioned studies clearly indicate that TCTP plays a critical role in the control of cell survival in vivo.
Besides its anti-apoptotic function, TCTP is required for cell growth and proliferation. For example, Drosophila TCTP was shown to control cell growth and proliferation by regulating GTPase activity of a Ras homologue, Rheb , although this point remains controversial, as previously discussed . In addition, TCTP regulates cell growth through its guanine nucleotide dissociation inhibitor activity for the elongation factors eukaryotic elongation factor 1A (eEF-1A) and elongation factor 1Bβ (EF1Bβ . TCTP is also a tubulin-  and calcium-binding  protein, serves as a substrate of polo-like kinase  and has properties of a histamine-releasing factor  or growth factor . These diverse functions of TCTP are likely to result from its specific association with various protein partners.
TCTP levels may vary considerably between various tissues and its synthesis is regulated at the transcriptional and post-transcriptional levels [21, 22], indicating involvement of tissue-specific factors. Analysis of the mouse Tctp (also known as Tpt1) gene promoter revealed that its expression is also regulated by cAMP . TCTP levels are, therefore, highly regulated in response to a wide range of extracellular signals and cellular conditions. Various stress conditions, such as starvation, heat shock, heavy metals, calcium stress or proapoptotic/cytotoxic signals can up- or downregulate TCTP levels, as previously reviewed . Furthermore, TCTP levels are regulated by the double-stranded RNA-dependent protein kinase (PKR) . In this context, we recently revealed that activation of PKR by pro-apoptotic stimuli results in downregulation of TCTP protein levels .
TCTP was first identified from tumour cells, but it has since been recognised that TCTP is not a tumour-specific protein, although its levels tend to be higher in tumours than in the corresponding normal tissue . There also seems to be a link between cancer and TCTP, since inhibition of TCTP results in suppression of the malignant phenotype . Intriguingly, reduced levels of TCTP have been detected in post-mortem brains from patients with Down’s syndrome and Alzheimer’s disease . It is plausible that diminished anti-apoptotic protection by TCTP is involved in these disorders.
Despite the many important functions attributed to TCTP, its role in pancreatic beta cells is not known to date. The current study identified TCTP as a novel glucose-regulated protein and investigated its role in glucose-regulated insulin secretion and cell survival.
All molecular biologicals were from Sigma (Poole, UK). Two-dimensional gel electrophoresis (2DGE) reagents and all secondary antibodies were from GE Healthcare (Chalfont St Giles, UK) and Invitrogen (Paisley, UK). Anti-mouse TCTP monoclonal antibody was from Stratech Scientific (Newmarket, UK). Monoclonal anti-phosphotyrosine and anti-phosphoserine antibodies were from Abcam (Cambridge, UK). TCTP-specific small interfering RNA (siRNA; sc-43450) and control scrambled RNAs were from Santa Cruz (Heidelberg, Germany). Rabbit polyclonal anti-extracellular signal regulated kinase (ERK)1/2 antibody was purchased from Cell Signaling (Hitchin, UK).
MIN6 and HIT-T15 beta cells were cultured as previously described . For glucose stimulation experiments, MIN6 cells were cultured for 12 h in DMEM containing 3 mmol/l glucose, which was then replaced by DMEM containing 3 or 25 mmol/l glucose for 24 h.
Isoelectric focusing and 2DGE
Cell homogenate was prepared by lysing the cells in RIPA buffer (138 mmol/l NaCl, 2.6 mmol/l KCl, 1.5 mmol/l KH2PO4, 6.3 mmol/l Na2HPO4, pH 7) containing 1% (vol./vol.) Igepal CA-630, 0.5% (wt/vol.) sodium deoxycholate, 0.1% (wt/vol.) SDS. Immobilised pH gradient (IPG) strips (24 cm) were rehydrated overnight and sample cup-loaded. Isoelectric focusing was performed for 1 h at 500 V with voltage increasing step-wise from 500 to 1,000 V, and finally to 8,000 V for 8 h. Strips were equilibrated for 15 min in LDS sample buffer (NuPAGE, Invitrogen, Paisley, UK) in the presence of 10% (vol./vol.) NuPAGE sample reducing agent. The samples were equilibrated for 15 min in LDS sample buffer (NuPAGE) containing 125 mmol/l iodoacetamide. Strips were transferred on to gels (Novex 4–12% Bis–Tris ZOOM gels; NuPAGE). The strips were overlaid with 0.5% (wt/vol.) agarose in running buffer (NuPAGE MOPS SDS) and gels run in a mini-cell device (XCell Surelock; Invitrogen).
Immunoblotting, immunocytochemistry and subcellular fractionation
These were performed as described earlier . To quantify nuclear localisation of TCTP in response to glucose, imaging was performed using the same confocal settings for all conditions and the intensity of nuclear staining was quantified by a software tool (Volocity; Perkin Elmer, Waltham, MA, USA). Three loading controls were used in this study. For the 2DGE and the corresponding 1DGE, the TCTP density was normalised to that of the total protein because ERK1/2 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) showed several spots on the 2DGE. GAPDH levels increased in response to glucose and thus could not be used as loading control in experiments where the glucose level changed. Thus, ERK1/2 was used as a loading control for islet proteins  and also for conditions where the glucose concentration was altered.
Detection of the phosphorylation state of TCTP
MIN6 beta cells were treated and proteins separated as described previously . The gels were co-stained with gel stains (Pro-Q Diamond Phosphoprotein and SYPRO Ruby; Invitrogen, Paisley, UK) according to the manufacturer’s instructions. The gels were imaged using a scanner (Typhoon 9210; GE Healthcare, Chalfont St Giles, UK) with excitation of 532 or 450 nm, and emission maximum of 580 or 610 nm for Pro-Q Diamond and SYPRO Ruby, respectively. Immunoblots were probed with phosphotyrosine- and phosphoserine-specific antibodies.
siRNA knockdown of Tctp expression and measurement of insulin secretion
This was carried out as described previously . For insulin secretion, cells at 96 h post-transfection were incubated for 1 h in KRB at 3 mmol/l glucose, followed by 30 min incubation in KRB at low (3 mmol/l) or high (30 mmol/l) glucose concentrations. Insulin was measured using a kit (Ultrasensitive Mouse Insulin ELISA; Mercodia, Uppsala, Sweden).
Measurement of apoptosis following Tctp siRNA transfection
Cells treated with siRNA were collected from the media and plate, and washed three times with PBS. The cell pellet was resuspended in 500 μl minimal medium (DMEM without FCS) and 25 μg/ml propidium iodide was added immediately before analysis. Fluorescence intensity of the propidium iodide bound to DNA was measured and apoptosis analysed using FACS analysis (FACSVantage SE FACS; Becton Dickinson, Oxford, UK) .
Isolation of islets of Langerhans and RT-PCR of Tctp
Islets were isolated as previously described . The study was conducted in accordance with the Principles of Laboratory Care. Total RNA was isolated using a reagent (TRI reagent, Sigma) according to the manufacturer’s protocol. RT-PCR was performed using primers corresponding to nucleotides 275-295 and 537-557 of the mouse Tctp (accession number NM_009429), respectively.
Fatty acid incubation
MIN6 or HIT-T15 cells were seeded on six-well plates and grown overnight. Palmitate was coupled to fatty acid-free BSA at a 5:1 molar ratio and added to the culture medium to final concentration of 0.5 mmol/l palmitate and 0.68% (wt/vol.) BSA. Cells were incubated for 24 h before use. Isolated islets were cultured for 16 h in DMEM containing 11 mmol/l glucose, hand-picked and incubated for 24 h in DMEM containing 3 or 17 mmol/l glucose in the presence or absence of 0.5 mmol/l palmitate.
Overproduction of TCTP and TUNEL assay
Cells were transfected with 0.5 μg pcDNA3 containing the coding region of full-length mouse Tctp  or pcDNA3 empty vector using lipofectamine (Invitrogen) as described previously . Cells at 24 h post-transfection were incubated with palmitate for 8 or 24 h. Cells were fixed and the TUNEL assay was performed according to the manufacturer’s instructions (Click-iT TUNEL Alexa Fluor 488; Invitrogen). The number of TCTP-overproducing cells and those positive for TUNEL staining were counted. Two controls were used: (1) cells transfected with pcDNA3; and (2) untransfected cells on the TCTP transfected plates in which TCTP overproduction was not visible.
Proteins were trypsin-digested using a robotic digester (Ettan digester; GE Biosciences, UK). Peptide mass fingerprints were acquired using Matrix-assisted laser desorption/ionisation time-of-flight mass spectrometer (MALDI-TOF; Waters Micromass, Waterloo, ON, Canada) with data acquisition and processing done with a software package (MassLynx; Waters, Elstree, UK). Database searching was performed by a software tool (ProteinLynx, Waters) using a peptide tolerance of 50 ppm.
TCTP levels are regulated by glucose
Post-translational modification of TCTP is regulated by glucose
MIN6 whole-cell lysates were separated by two dimensional SDS-PAGE, using narrow range (pH 3–5) IPG strips to increase the resolution and improve the separation of the two proteins recognised by the TCTP antibody. Using anti-phosphoserine and anti-phosphotyrosine antibodies, we observed that the 23 kDa TCTP was apparently dephosphorylated on these residues in response to glucose stimulation (Fig. 3). The presence of a phosphorylated TCTP isoform was confirmed by phosphostaining (ProQ Diamond) (Fig. 3) and subsequent identification of the protein band with quadrupole time-of-flight (Q-TOF) mass spectroscopy analysis. We also investigated the potential glycosylation of TCTP. However, the monoclonal antibody against O-GlcNAc modification or the glycoprotein stain Emerald 488 (Pro-Q) produced very high background and many non-specific staining signals. Thus, the glycosylation state of TCTP proteins could not be resolved convincingly. Nevertheless, regulation of TCTP production by glucose and dephosphorylation on serine and tyrosine residues were clearly demonstrated.
Increased nuclear localisation of TCTP in response to glucose
Reduction of TCTP protein production results in enhanced beta cell death
Palmitate reduces TCTP protein production through mechanisms involving endoplasmic reticulum stress
Increased TCTP protein production protects cells from apoptosis
To better understand the possible mechanism by which increased TCTP protein levels reduce cell death, we next tested whether a higher proportion of TCTP becomes associated with the mitochondria under this condition. In non-stimulatory glucose condition only a very small amount of TCTP was detectable in the mitochondrial fraction, whereas at stimulatory glucose levels the amount of TCTP found in this subcellular fraction was clearly increased (Fig. 7c). This supports a model, according to which TCTP anchors to the mitochondrial membrane and antagonises the action of the pro-apoptotic protein BAX, which is involved in regulating the membrane permeability .
To contribute to the understanding of glucose regulation in pancreatic beta cells, we investigated the differential regulation of proteins in response to high vs low glucose concentrations. Using a proteomics approach, we identified 11 out of 84 proteins that are differentially regulated (Fig. 1, ESM Tables 1–3). Of these proteins, we initially selected TCTP for a more detailed investigation.
Our study demonstrates that TCTP protein levels and subcellular distribution are regulated by glucose in pancreatic beta cells. Since increased fatty acid circulation is a major factor in the development of type 2 diabetes and fatty acids have been shown to be toxic to beta cells [34, 35, 36, 37], we also investigated the effect of a fatty acid on TCTP protein levels in beta cells. Palmitate downregulated TCTP protein levels and resulted in increased cell death (Fig. 6a–d). Thus, the cytotoxic effect of palmitate is, at least in part, due to reduced production of this anti-apoptotic protein. In contrast, overproduction of TCTP protein protected beta cells from apoptosis (Fig. 7a, b). Our results thus imply that TCTP levels influence the sensitivity of the beta cell to apoptosis. TCTP levels have been shown to be highly regulated in response to a wide range of extracellular signals, as reviewed by others [6, 21, 22]. However, to our knowledge, this is the first demonstration of regulation of TCTP levels in response to alterations in glucose or fatty acid concentrations.
Protein synthesis in beta cells is highly regulated in response to alterations of glucose concentrations. In particular, mechanisms of the unfolded protein response (UPR), a physiological ER-stress response, are involved in this regulation. This includes activation of the PKR-like ER kinase (PERK) and subsequent phosphorylation of initiation factor eIF2α, leading to (local) inhibition of protein synthesis. At low glucose concentrations, PERK is activated, resulting in increased eIF2α phosphorylation and inhibition of ER protein (and therefore proinsulin) synthesis [38, 39]. We have previously demonstrated that TCTP synthesis is downregulated through phosphorylation of eIF2α by PKR in cellular stress conditions [11, 24]. Although we did not specifically address the role of PERK in these studies, both kinases are probably involved in TCTP regulation, as both are activated upon ER-stress . We conclude that the glucose-dependent regulation of TCTP synthesis in beta cells observed in this study (Fig. 2) is also likely to be mediated through a mechanism involving eIF2α phosphorylation.
While mechanisms of the UPR are involved in physiological regulation of beta cells through glucose, they may also be activated under chronic stress conditions, such as elevated levels of NEFA, which contribute to beta cell dysfunction in type 2 diabetes. NEFA, such as palmitate, trigger ER stress [34, 35, 36, 37] and eventually apoptosis in cultured beta cells in vitro, as reviewed by others [41, 42]. They also induce apoptosis in primary rodent and human islets of Langerhans . In beta cells, palmitate has been demonstrated to activate PERK and phosphorylation of eIF2α, resulting in inhibition of translation initiation [35, 36, 43]. In a recent study, we demonstrated downregulation of TCTP levels in mouse embryo fibroblasts under some, but not all ER stress conditions . TCTP was particularly downregulated in Ca2+ stress conditions (including thapsigargin treatment) in a manner dependent on active PKR and eIF2α phosphorylation. This is consistent with our observation that TCTP is downregulated in beta cells under stress induced by palmitate or thapsigargin (Fig. 6). Downregulation of anti-apoptotic proteins is an important prerequisite for apoptosis to occur, so it is not surprising that TCTP  and MCL-1  have been found to be downregulated in Ca2+/ER stress conditions in a PKR-dependent manner. Since palmitate has earlier been shown to result in activation of PERK in beta cells [35, 36, 43], we conclude that downregulation of TCTP by palmitate (Fig. 6) is mediated through PERK activation.
Our studies have also shown that overproduction of TCTP can delay apoptosis induced by thapsigargin, tunicamycin and etoposide  or palmitate (Fig. 7a). Conversely, downregulation of TCTP protein production has been reported to lead to apoptosis [10, 13, 14], consistent with the observations obtained here in Tctp knockdown experiments on beta cells (Fig. 5b). Palmitate is known to be cytotoxic to beta cells in obesity-associated animal models of diabetes, as well as in normal beta cells [34, 35, 36, 37]. We propose that these cytotoxic effects are at least in part mediated by the inhibition of production of anti-apoptotic proteins such as TCTP.
TCTP has been described as a cytosolic protein , but nuclear localisation has also been reported [12, 45]. Under low glucose concentrations, we observed largely cytosolic TCTP staining in beta cells, with very little staining in the nucleus. However, under high-glucose conditions we noticed a significant TCTP immunostaining in the nucleus, in addition to the cytosolic staining (Fig. 4a). Similarly to our finding, other recent studies have shown that TCTP can be transported to the nucleus under certain conditions . Rid et al.  reported that under oxidative stress conditions TCTP translocates to the nucleus. There are also indications that TCTP might be involved in transcriptional regulation of specific genes in T lymphocytes  and in early development [47, 48]. Our current study indicates that TCTP becomes dephosphorylated on serine and tyrosine residues in response to glucose stimulation, which might be important for this nuclear translocation, as reported in other cases [31, 32, 33].
Since TCTP is an anti-apoptotic protein, we also attempted to investigate its potential co-localisation with mitochondria in beta cells. However, because TCTP is still abundantly present in the cytosol, giving rise to intense cytosolic staining, and because these cells were only poorly spread, it was not possible to localise TCTP to any specific organelles using immunocytochemistry. Instead, we used subcellular fractionation, the results of which indicated that a proportion of TCTP becomes associated with the mitochondria-enriched fraction at high vs low glucose condition (Fig. 7c). It has been demonstrated that TCTP interacts with the anti-apoptotic proteins MCL-1 and BCL-XL in vitro [8, 9]. Immunofluorescence data have also suggested that TCTP co-localises with BCL-XL  and MCL-1  in vivo, and is therefore likely to act, like other key regulators of apoptosis, at the mitochondria. However, the interpretation of co-localisation studies [8, 9] is rather complicated because, as mentioned above, TCTP is highly abundant in the cytosol. A more recent study has demonstrated that the anti-apoptotic function of TCTP takes place, at least in part, at mitochondria, with TCTP inhibiting the function of BAX . Our investigations (Fig. 7c) suggest that, in beta cells, TCTP partially translocates to the mitochondria in response to glucose, where it is likely to act in a similar fashion.
In summary, we have provided evidence that, in pancreatic beta cells, TCTP is positively regulated by glucose and negatively by pro-apoptotic stimuli, such as palmitate. It is involved in the protection of beta cells against apoptosis. Further studies are required to determine the precise mechanisms of the glucose-dependent regulation of TCTP and its translocation to the nucleus, as well as the significance of this translocation for its anti-apoptotic function. Further studies are also required to elucidate regulation of TCTP by other pro-apoptotic stimuli in beta cells, as well as the importance of TCTP in animal models of type 2 diabetes.
We thank J. Slinn and M. Lewis (Centre for Research in Biomedicine, Faculty of Health and Life Sciences, University of the West of England, Bristol, UK) for their technical assistance and advice on mass spectroscopy analysis. This study was supported by grants from the Wellcome Trust, the Biotechnology and Biological Sciences Research Council and the Medical Research Council to A. Varadi. S. Lajus was supported by a Marie Curie Intra European Fellowship within the 7th European Community Framework Programme.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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