Molecular mechanisms of tungstate-induced pancreatic plasticity: a transcriptomics approach
© Altirriba et al; licensee BioMed Central Ltd. 2009
Received: 7 January 2009
Accepted: 28 August 2009
Published: 28 August 2009
Sodium tungstate is known to be an effective anti-diabetic agent, able to increase beta cell mass in animal models of diabetes, although the molecular mechanisms of this treatment and the genes that control pancreas plasticity are yet to be identified. Using a transcriptomics approach, the aim of the study is to unravel the molecular mechanisms which participate in the recovery of exocrine and endocrine function of streptozotocin (STZ) diabetic rats treated with tungstate, determining the hyperglycemia contribution and the direct effect of tungstate.
Streptozotocin (STZ)-diabetic rats were treated orally with tungstate for five weeks. Treated (STZ)-diabetic rats showed a partial recovery of exocrine and endocrine function, with lower glycemia, increased insulinemia and amylasemia, and increased beta cell mass achieved by reducing beta cell apoptosis and raising beta cell proliferation. The microarray analysis of the pancreases led to the identification of three groups of differentially expressed genes: genes altered due to diabetes, genes restored by the treatment, and genes specifically induced by tungstate in the diabetic animals. The results were corroborated by quantitative PCR. A detailed description of the pathways involved in the pancreatic effects of tungstate is provided in this paper. Hyperglycemia contribution was studied in STZ-diabetic rats treated with phloridzin, and the direct effect of tungstate was determined in INS-1E cells treated with tungstate or serum from untreated or treated STZ-rats, observing that tungstate action in the pancreas takes places via hyperglycemia-independent pathways and via a combination of tungstate direct and indirect (through the serum profile modification) effects. Finally, the MAPK pathway was evaluated, observing that it has a key role in the tungstate-induced increase of beta cell proliferation as tungstate activates the mitogen-activated protein kinase (MAPK) pathway directly by increasing p42/p44 phosphorylation and indirectly by decreasing the expression of raf kinase inhibitor protein (Rkip), a negative modulator of the pathway.
In conclusion, tungstate improves pancreatic function through a combination of hyperglycemia-independent pathways and through its own direct and indirect effects, whereas the MAPK pathway has a key role in the tungstate-induced increase of beta cell proliferation.
The endocrine pancreas is continually remodelled  in a dynamic process involving the death and regeneration of beta cells. Though several mechanisms have been implicated in adult beta cell maintenance and renewal , it has been demonstrated that the proliferation of differentiated beta cells is the major mechanism for the maintenance of adult beta cell mass . Nevertheless, it has recently been shown that endogenous progenitors can participate in the increase in beta cell mass in adult mice .
Studies in several animal models of diabetes have shown sodium tungstate to be an effective anti-diabetic agent, able to reverse hyperglycemia [5–9]. Tungstate treatment normalizes liver glucose metabolism in streptozotocin (STZ) and Zucker Diabetic Fatty (ZDF) rats [5, 7], improves beta cell function in neonatal streptozotocin (nSTZ) rats , and also decreases hypertriglyceridemia in ZDF-rats . In nSTZ-rats, tungstate not only restores glucose-induced insulin secretion, but also completely regenerates beta cell mass, leading to normoglycemia in animals even after withdrawal of the treatment . Sodium tungstate has also been shown to exert insulin-mimetic effects in isolated hepatocytes  and to increase insulin content and enhance insulin secretion in the presence of glucose and various segretagogues in an insulinoma cell line and the perfused pancreas [11, 12].
The aim of this study is to unravel the molecular mechanisms of tungstate action in the pancreas. Streptozotocin is a diabetogenic compound and is known to specifically destroy beta cells in the pancreas, obtaining a diabetic animal model with impaired endocrine and exocrine function and reduced beta cell mass. In this model, we identified the pancreatic gene expression changes induced by the streptozotocin damage and the partial recovery of function after treatment with tungstate. The data presented here show that tungstate treatment modifies the expression of a set of genes which participate in the tungstate-induced pancreatic plasticity, with a recovery of exocrine function and a partial recovery of endocrine function and beta cell mass. Moreover, we demonstrate that most of these effects are independent of the recovery of hyperglycemia and due to direct and indirect effects of tungstate in the pancreas. Further study of these pathways may lead to the discovery of new strategies for modifying pancreatic plasticity.
Effects of tungstate treatment on blood and beta cell parameters
Gene expression profile of diabetic treated animals
In Figure 2b, the 282 genes differentially expressed due to diabetes are grouped according to their function [for a complete list, see Additional file 1]. The analysis of the arrays from DT animals identified 88 differentially expressed genes (see Figure 2c for functional clustering, and Additional file 2 for the complete list). The majority of these genes (70%) represented genes whose expression was restored, in varying degrees, by the tungstate treatment. Among these genes are the ones involved in metabolism, protein modification, oxidative stress, and so on, showing that the treatment acts on several pathways which lead to the recovery of pancreatic function. Only 28 of the 88 genes were differentially expressed in DT animals alone [see Additional file 3].
Validation of the array results
Tungstate effects are independent of glycemia normalization
Hyperglycemia has a critical impact on beta cell function  and gene expression . To assess the involvement of the decrease in the hyperglycemia on tungstate effects, we decided to explore whether the gene expression changes induced by the treatment were just the consequence of the decrease in glycemia observed in the diabetic treated animals. To do so, diabetic rats were treated with phloridzin, which selectively inhibits active glucose reabsorption in the kidneys and, as opposed to insulin administration, leads to a decrease in glycema without any direct effect on pancreas function. Phloridzin administration led to a significant decrease in glycemia, though healthy values were not attained; interestingly, the levels reached were quite similar to those observed in DT animals (Figures 3b and 1a). Analysis of the expression of the genes validated by Real Time PCR showed that most did not present significant differences between the phloridzin administrated animals and their control counterparts (Figure 3c). Nevertheless, Sel1h and insulin expression increased significantly (insulin: 1.82 ± 0.32 fold increase, Sel1h: 1.45 ± 0.18 fold increase, p < 0.05 vs. untreated animals), although this increase was lower than the one observed in the tungstate-treated diabetic rats (insulin: 4.49 ± 1.10 fold increase, Sel1h: 4.21 ± 0.31, p < 0.001 vs. DU). Nuclear protein 1 (Nupr1), which has been shown to be regulated by glucose levels , was included as a positive control, and, as expected, it was significantly reduced in phloridzin-treated animals. Therefore, we conclude that tungstate effects on gene expression are independent of the decrease in glycemia.
Tungstate increases beta cell proliferation indirectly through the modification of the serum profile
MAPK pathway is involved in tungstate induced beta cell proliferation
Among several other cellular functions, the MAPK pathway plays a key role in cell replication . One of the tungstate-specific genes that we have analyzed is Rkip, which is involved in the fine tuning of this important pathway in the islets through the inhibition of Raf-1 . This gene was increased in the diabetic untreated animals compared to untreated healthy rats (inhibiting the MAPK p42/44 pathway), and the treatment returned its levels to normal (re-establishing MAPK p42/44 signalling) (Figure 3a). Moreover, previous studies  have demonstrated that sodium tungstate is able to activate the MAPKp42/44 pathway by increasing p42/44 phosphorylation in hepatocytes. Therefore, we decided to further evaluate the role of the MAPK pathway in tungstate-induced beta cell replication. We observed that tungstate was able to stimulate the MAPK pathway in INS-1E (Figure 4c and 4e). Similarly, the serum from the diabetic treated animals was also able to enhance the MAPK pathway more strongly than the serum from the untreated animals (notice the different scales used in Figures 4e and 4f); in addition, the activation of the pathway lasted longer (Figures 4d and 4f). This MAPKp42/44 activation pattern mimics the findings observed with the INS-1E proliferation in the presence of tungstate and serum from the different animals, showing also that serum from diabetic treated animals is a more potent proliferative agent and activator of the MAPK p42/44 pathway. Since it seemed that the stimulation of beta cell replication was linked to MAPK activation, we decided to culture INS-1E cells with PD98059, a MAPK/ERK kinase 1 (Mek1) inhibitor . In the presence of PD98059, the serum from the treated animals induced a lower stimulation of beta cell replication (62% reduction, p < 0.05, n = 6), but no significant effect was seen in the serum from the untreated animals (8% reduction, p = n.s., n = 6). These data suggest that in order to exert its effects on beta cell replication, tungstate has to activate the MAPK pathway by both short-term (direct activation) and long-term mechanisms (normalization of Rkip expression), probably through an indirect mechanism involving either tungstate metabolization or the stimulation of some beta cell trophic factor(s).
This paper focuses on the molecular mechanisms by which tungstate administration modulates pancreatic plasticity in diabetic animals. We selected the streptozotocin rat model, which shows a high degree of pancreatic damage at both endocrine and exocrine levels . In this situation, tungstate administration partially restores glycemia, insulinemia and amylasemia. Moreover, the morphological results clearly demonstrate that treatment increased beta cell mass in the pancreas and increased insulin and Pdx-1 positive cells in the islets. This increased beta cell mass can be ascribed, at least in part, to a combination of a decrease in beta cell apoptosis and an increase in islet proliferation.
In agreement with the metabolic phenotype, the pancreatic microarray analysis found no differences in the gene expression pattern between HU and HT animals, but it showed a very different pattern in DU animals as compared with both healthy samples. These changes are found not only in genes linked to endocrine function but also in those linked to exocrine function, probably due to the toxic effects of STZ. Finally, DT animals presented a specific pattern which was different from both DU animals and healthy animals, due to the effects of sodium tungstate on the diabetes background.
In relation to the recovery of exocrine function, we should mention the possible role of Xbp1. This gene plays a key role in the unfolded protein response , and Xbp1 knockout animals display abnormalities exclusively in secretory organs such as the exocrine pancreas, which lead to early postnatal lethality . In our model, we found Xbp1 to be decreased in the DU animals and increased in the DT animals concomitantly to the recovery of the amylase expression, a finding that stresses the important role of this gene in the correct function of the exocrine machinery.
Several genes whose expression was modified by tungstate administration to diabetic animals may be responsible for the recovery of pancreatic function. Those genes are involved in different pathways which control a wide range of physiological functions, covering growth and differentiation to morphogenesis and angiogenesis. Therefore, it is probably the combination of tungstate effects upon all these pathways that leads to the recovery of pancreatic function. Since we performed microarrays using the total pancreas, we cannot identify the exact sites of these changes (exocrine, endocrine or ducts). Nevertheless, it seems that their modulation may contribute to the pancreatic regeneration observed in the diabetic treated animals. Fgf13 is one of the genes modified in the diabetic treated animals. Fgf13 binds to islet-brain 2, recruiting p38δ and increasing the activity of this kinase . Interestingly, it has already been shown that tungstate increases the phosphorylation of p38 in both MIN6 beta cells  and islets of nSTZ-rats , which may lead to the improvement observed in the pancreatic beta cell population. Another modified gene was Tspn8, which may exert tumor-promoting activities by increasing cell motility and by inducing angiogenesis . Therefore, bearing in mind that STZ damage provokes severe microcirculatory disturbances within pancreatic islets , we propose that an increase in the Tspn8 expression may improve pancreas revascularization and enhance islet function. Finally, other interesting genes are Tgfb3, Usag-1 and Sel1h. Tgfb3 plays a key role in complex processes including epithelial to mesenchymal transition , which is relatively important in pancreas regeneration [29, 30]. Usag-1 modulates Wnt and Bone Morphogenic Protein signaling [31–33] in different ways. Sel1h has been implicated as a Notch inhibitor in exocrine  and endocrine replication  and modifies the expression of genes involved in cell-matrix interactions and in the cell cycle . Although the exact role of these genes in pancreatic regeneration has not been investigated in detail, we hypothesize that the combined action of these multiple pathways leads to the regeneration of the pancreas observed in the DT rats.
Although many pathways are involved in tungstate action, the results clearly show that the MAPK pathway plays a key role. The array analysis showed normalization in Rkip expression in the DT animals. This protein binds to v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1), leading to the blockage of Mek and extracellular-signal-regulated kinase 1 (Erk) activation. It has been shown that, in the pancreas, Rkip localizes specifically in the islets and is involved in the regulation of beta cell growth. When this gene is up-regulated, as in the case of the DU animals, it acts as a brake on the MAPK signaling and beta cell proliferation . The normalization of Rkip in the DT rats would permit the unblocking of the MAPK signaling and thus allowing the proliferative signals through this pathway. In the liver, it has been shown that tungstate enhances glycogen synthesis through MAPK activation . Here we demonstrate that tungstate activates MAPK both directly and indirectly and is crucial for the tungstate-induced increase observed in beta cell replication. On the one hand, tungstate enhances MAPK phosphorylation; on the other, it normalizes Rkip expression which also leads to an increase in the MAPK pathway tone. The combination of these two mechanisms is the cause of the increased beta cell proliferation observed in our study.
In summary, tungstate is able to increase beta cell mass and recovers the exocrine and partially endocrine function in STZ-rats. These improvements in pancreatic function require the combined action of several pathways, such as Wnt, TGF-b, Notch and MAPK. The last of these is clearly involved in the increase in beta cell mass. This study identifies several proteins that may play a key role in the improvement of pancreatic plasticity. Further studies will help us to identify their precise role in the pancreas.
Animals, induction of diabetes and tungstate treatment
Principles of laboratory animal care were followed (European and local government guidelines) and protocols were approved by the Animal Research Committee of the University of Barcelona. Diabetes induction and tungstate treatment have been described previously . Diabetes was induced in adult male Wistar rats (225 g) obtained from Charles River (Wilmington, MA, USA) by an intraperitoneal STZ (Sigma, St Louis, MO, USA) injection (70 mg/Kg). At the beginning of the experiment, both diabetic and healthy animals were randomly divided into two groups. For 5 weeks, treated rats received ad libitum a solution of 2 mg/ml sodium tungstate (Carlo Erba, Rodano, Italy) in deionized water; whereas the untreated rats received deionized water alone (daily sodium tungstate intake: healthy 71 mg, diabetic 110 mg). Therefore, the study had 4 experimental groups (healthy untreated [HU], healthy treated [HT], diabetic untreated [DU] and diabetic treated [DT]). Glycemia was measured every 5 days. At the end of the experiment, blood was collected, and serum was obtained. For islet studies, islets were isolated by collagenase digestion, as previously described .
In an additional group of animals, diabetes was induced by STZ injection, as described in the previous section. Once the hyperglycemia was confirmed, these diabetic animals were subdivided into two groups, one group (control) which received the vehicle used to dissolve phloridzin; and the other group (treated) which received phloridzin as previously described : phloridzin (Sigma) was dissolved (40%) in propylene glycol (Sigma) and injected intraperitoneally every 8 h during 1 week at a dose of 2 g/Kg body weight per day. Phloridzin treated rats also underwent a caloric restriction of 0.1 g chow diet/Kg body weight per day.
Insulinemia and amylasemia
Insulinemia was measured by radioimmunoassay (CIS, Gif-sur-Yvette, France), and amylasemia was determined in an Advia Analyzer 2400 (Siemens Medical Solutions Diagnostics, Munich, Germany)
Immunohistochemical studies and beta cell analysis
The whole pancreas was removed, fixed and embedded in paraffin. Serial sections of pancreas (5 μm) were obtained from three different levels. Beta cells were located using a modified avidin-biotin-peroxidase method . The morphometric analysis was performed using OPTIMAS™ (Bioscan Incorporated, Edmonds, WA, USA) software. We also stained for pancreatic duodenal homeobox-1 (Pdx-1) (kindly provided by Dr. C. Wright), quantifying the percentage of Pdx-1-labeled cells within the islet cells (not less than 1000 cells were counted).
Cell replication rates
Sequential-dual staining for proliferating cell nuclear antigen (PCNA) (Sigma) and insulin (Dako, Glostrup, Denmark) was performed. Replication rate was quantified and expressed as the percentage of PCNA-labeled cells among the total beta cells counted (not less than 3000 each). Immunohistochemical stainings have been validated in previous studies [39, 40].
To identify apoptotic bodies, we used the propidium iodide technique . Deparaffinized and rehydrated sections were incubated for 30 minutes in a solution of propidium iodide (4 μg/ml, Sigma) and RNAse A (100 μg/ml, Sigma). For the quantitative evaluation, positively labeled apoptotic endocrine cells were counted, and the number of apoptotic cells was expressed as the percentage of the total number of beta cells counted.
RNA was isolated from fresh total pancreas using the RNAgents Total RNA Isolation System (Promega, Madison, WI, USA). Islet RNA was isolated with the RNeasy Mini Kit (Qiagen, Venlo, The Netherlands). RNA integrity was analyzed using a Lab-on-a-chip in a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Total RNA from 3 rat pancreases of the same experimental group was pooled in equal proportions and hybridized to a Rat GeneChip RAE230-A (Affymetrix, Santa Clara, CA, USA). In total, 12 microarrays were hybridized using 3 different pools of each experimental group. The data were deposited in NCBIs Gene Expression Omnibus with the accession number GDS1883. Several quality controls were performed, as described previously  [see Additional file 5, 6, 7 and 8], and analyzed, as described in Additional file 9, using Affy [42, 43], AffyPLM  and LIMMA  packages from Bioconductor  on R language [47, 48] and dChip software .
Values for clustering were obtained after standardizing the gene values: the mean value of each gene was subtracted from the value of this gene in each array and was divided by the standard deviation of this gene in all the arrays. Clustering was performed with a distance metric of 1 – correlation, centroid linkage method and gene ordering by cluster tightness
Real Time PCR
Total RNA was retrotranscripted with SuperscriptIII (Invitrogen, Carlsbad, CA, USA). Real Time PCR was carried out in a 7900 HT Real Time System (Applied Biosystems, Foster City, CA, USA) using SYBR Green fluorophor. The primers used are described in Additional file 10. A standard curve of each primer set was generated from serial dilutions of cDNA. The PCR products were verified by way of dissociation curve analysis using SDS software (Applied Biosystems). Expression levels obtained were normalized with a housekeeping gene (TATA box binding protein) and were scaled to the mean of the values of the same gene found in the healthy untreated animal.
Western blotting was performed as described previously , using a specific antibody against phospho-p42-p44 MAPK (Thr202/Tyr204; Cell signaling, Danvers, MA, USA). Membranes were then stripped and incubated with anti-p42-p44 MAPK (Cell signaling). Intensity values were obtained with Image Gauge 4.0 software (Fujifilm, Valhalla, NY, USA). These values were divided by the value obtained at time 0, in order to normalize the data and permit comparisons among experiments performed on different days. Finally, data was log 2 transformed in order to obtain additive normally distributed noise and to stabilize variance .
INS-1E cells (kindly provided by Dr P. Maechler) were maintained as described elsewhere . For serum proliferation studies, two days after seeding, the medium was substituted by a medium containing RPMI1640 (Biosera, Ringmer, UK), BSA 0.1%, glucose 5 mmol/l and sodium tungstate at 100 μmol/l (Carlo Erba) or 10% of the serum from the animals. 24 hours later, hydroxyurea 12.5 mmol/l (Sigma) was added to the medium. 24 hours later, the medium was removed, and wells were cleaned with DPBS (Sigma). A medium with the initial composition supplemented with methyl-3H-Thymidine 1 μCi (initial specific activity 74 GBq/mmol) (GE Healthcare, Fairfield, CT, USA) was added to each well for 4 hours. Plates were then frozen and defrosted, cells were harvested with a Cell Harvester, and cpm was measured with a 1205 BetaPlate liquid scintillation counter (Perkin Elmer, Waltham, MA, USA).
Two days after seeding INS-1E cells, in order to decrease basal phosphorylation levels, the medium was substituted by RPMI1640 without any supplement. Eight hours later, the medium was substituted by RPMI1640 complemented with 10% of the serum from animals of the different experimental groups or sodium tungstate (100 μmol/l). After 0, 5, 15, 30 and 60 minutes, the plates were rinsed with DPBS and frozen.
Quantitative data are expressed as means ± SEM. The statistical significance was determined by Student's t-test (when only two groups were analyzed: glycaemia of the phloridizin treated animals, Real-time PCR values of the phloridizin treated animals and tungstate treated cells replication) and ANOVA (when more than two groups were analyzed: the rest), with a Tukey's Post-Hoc test used to find the groups responsible for the statistical significance.
This study was supported by grants SAF 2006/07382 (Ministerio de Ciencia y Tecnología, Spain), FIS 02/0483, FIS 04/2553 (Ministerio de Sanidad y Consumo, Spain), SGR 2001/00378 (Generalitat de Catalunya, Spain), and by the Plan Nacional de Investigación Científica (I+D+I), Ingenio 2010, and Programa Consolider. CIBER de Diabetes y Enfermedades Metabólicas Asociadas is an initiative of ISCIII (Ministerio de Ciencia e Innovacion). J.A. was recipient of a grant from IDIBAPS and B.N. from the Generalitat de Catalunya. We thank M. Julià. K. Katte and M. Maudsley for their assistance.
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