Inhibition of GSK3 Promotes Replication and Survival of Pancreatic Beta Cells
2007; Elsevier BV; Volume: 282; Issue: 16 Linguagem: Inglês
10.1074/jbc.m609637200
ISSN1083-351X
AutoresRainer Mußmann, Marcus Geese, Friedrich Harder, Simone Kegel, Uwe Andag, Alexander Lomow, Ulrike Burk, Daria Onichtchouk, Cord Dohrmann, Matthias Austen,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoRecent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2–3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies. Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2–3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies. 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Using potent and specific small molecule GSK3 inhibitors, we found that the inactivation of GSK3 protects beta cells against death induced by high concentrations of glucose and the saturated fatty acid palmitate. In addition, small molecule GSK3 inhibitors robustly stimulate beta cell proliferation. These results suggest that GSK3 plays a key role in the regulation of beta cell mass and is a target for beta cell regenerative therapies. Cell Culture—INS-1E cells were maintained in culture medium (RPMI 1640 containing 11 mm glucose, 5% fetal calf serum, 10 mm HEPES, 50 μm 2-mercaptoethanol, 1 mm sodium pyruvate) and cultivated at 37 °C, 5% CO2 in a humidified atmosphere as described (42Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (471) Google Scholar), except as otherwise stated. Cells were seeded into tissue culture flasks at a density of 2 × 104 cells/cm2 6–8 days before treatment. During the growth period, the medium was changed twice. Isolation of Rat Islets—Islets were isolated by the standard Liberase digestion method from rat pancreata (Liberase™ CI enzyme blend BMB, catalog number 1814-435; Roche Applied Science) and cultured as described (43Brun T. Franklin I. St-Onge L. Biason-Lauber A. Schoenle E.J. Wollheim C.B. Gauthier B.R. J. Cell Biol. 2004; 167: 1123-1135Crossref PubMed Scopus (123) Google Scholar). In brief, 10 ml of ice-cold Liberase solution (Roche Applied Science) was injected into the pancreas via the common bile duct. After dissection, the pancreas was incubated for 40 min at 37 °C and then further dissociated by repeated pipetting using a 10-ml pipette. Islets were purified by a Ficoll density gradient centrifugation and were manually picked using a stereomicroscope. Islets were placed in bacteriological wells, and factors were administered as indicated. For quantitative reverse-phase PCR, islets were harvested in Trizol™ and immediately transferred to dry ice. RNA was isolated according to common procedures. Viability Assay, Cell Number Determination, Caspase Activity Assay, and DNA Fragmentation Assay—INS-1E cells were seeded at a density of 1 × 104 cells/96-well plate (black 96-well tissue culture plates; Falcon catalog number 353948) and grown in 100 μl of culture medium/well supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin for 3 days. The metabolic activity or viability of INS-1E cells was assessed by mitochondric reduction of the nontoxic dye Alamar Blue (Biosource catalog number DAL1025). The dye was added to the cells 4 h before read-out according to the manufacturer's guidelines. For the determination of relative cell numbers, INS-1E cells were maintained for 24 h in 100 μl of starvation medium (culture medium with only 5 mm glucose, 1% fetal calf serum) before the test compounds were added for an additional 4 days. Then the tissue culture plates were washed one time with 200 μl of phosphate-buffered saline and frozen at -80 °C for at least 1 h. Cell number was measured by staining of cellular DNA with CyQuant dye (CyQuant cell proliferation assay kit; Molecular Probes, Inc., catalog number C-7026), which becomes fluorescent when bound to DNA. Fluorescence was measured after 30 min of incubation using the FLUOstar Optima reader from BMG Labtechnologies. Glucolipotoxicity-mediated apoptosis was induced by the addition of a combination of bovine serum albumin-coupled palmitate (0.3 mm palmitate, bovine serum albumin (palmitic acid sodium salt/fatty acid-free bovine serum albumin, 6:1)) and glucose (25 mm) to the starvation medium followed by a 24-h incubation period. Test compounds were added 1 h prior to the addition of apoptosis-inducing factors. Caspase activity was quantified by an enzymatic assay (homogeneous caspase assay; Roche Applied Science catalog number 03 005 372 001). Caspase activity was measured 3 h after the addition of caspase substrate according to the guidelines of the manufacturer. The frequency of apoptosis was also monitored by the determination of mono- and oligonucleosomes in the cytoplasmic fraction of cell lysates using the cell death detection enzyme-linked immunosorbent assay kit (Roche Applied Science catalog number 1774425). The assay is based on a quantitative sandwich enzyme immunoassay principle using mouse monoclonal anti-histone and anti-DNA peroxidase antibodies. The relative frequency of apoptosis was photometrically determined by measuring the peroxidase activity of the immunocomplexes at 405 nm. Fluorescence or photometric measurements were carried out with the FLUOstar Optima reader from BMG Labtechnologies. In Vitro Beta Cell Proliferation Assay—Freshly isolated islets were cultured in vitro with or without the addition of factors of interest for 48 or 72 h. Following the culture period, the islets were dispersed gently by titration in Ca2+- and Mg2+-free phosphate-buffered saline. The resulting single cell suspension was applied to adhesive slides at 3000–6000 cells/well (adhesion slides/Fa Superior Marienfeld REF 09 000 00). The adherent islet cells were fixed and stained by standard immunofluorescence techniques for C-peptide, a fragment of proinsulin, and Ki-67 a marker of proliferating cells. Nuclear DNA was stained with 4′,6-diamidino-2-phenylindole (Molecular Probes). An Olympus microscope equipped with an automatic image acquisition device (Olympus) was used for counting of C-peptide-positive beta cells. Proliferating C-peptide/Ki-67 double positive beta cells were counted manually. The fraction of proliferating beta corresponds to the percentage of Ki-67 double positive cells of all C-peptide-positive cells. Using this assay, the growth-promoting effects of a number of known beta cell mitogens, including prolactin, GIP, hepatocyte growth factor, epidermal growth factor, growth hormone, exendin-4, betacellulin, fibroblast growth factor-2, and activin on rat beta cells were detectable (data not shown). Data generated with prolactin and GIP are presented in Fig. 4. BrdUrd Labeling and Detection Assay—INS-1E cells were seeded in 96-well culture plates and cultured as described above for the caspase activity assay. BrdUrd labeling and detection was carried out with a commercially available kit (cell proliferation enzyme-linked immunosorbent assay, BrdUrd (chemiluminescence) Roche Applied Science catalog number 1 669 915) according to the manufacturer's instructions. In brief, 24 h after switching to starvation medium, test compounds were added, and cells were cultured for an additional 24 h. BrdUrd labeling solution was added to the medium for the last 4 h before the cells were fixed using FixDenat solution and incubated with monoclonal anti-BrdUrd-POD antibodies. After substrate solution was added to each well, the light emission was measured in a microplate luminometer using the Analyst™ HT detection system from LJL Biosystems Inc. RNA Interference, Western Blotting, and Proliferation Assay— For Western blotting, INS-1E cells were seeded in 12-well plates at a density of 2 × 105 cells/well and cultured overnight before transfection. Cells were transfected with 6 μl of HiPerFect Transfection Reagent (Qiagen) and mixed with 5 nm siRNA duplexes (Qiagen). For quantitative reverse transcription-PCR, cells were harvested in Qiagen lysis buffer 72 or 140 h after transfection. For Western blot analysis, cells were harvested in a standard protein lysis buffer. Total protein was separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane. Blots were developed using the chemiluminescence detection system SuperSignal West Dura from Pierce (product number 34075). The following antibodies were used: mouse monoclonal anti-GSK3α and -β (Calbiochem catalog number 368662) at a 1:1000 dilution, mouse monoclonal anti-α-tubulin (Sigma catalog number T6557) at a 1:10,000 dilution, and as a secondary antibody horseradish peroxidase-conjugated goat anti-mouse antibody (Pierce product number 34075) at a 1:5000 dilution. For the determination of the proliferation rate of INS-1E, cells were seeded in 96-well plates at a density of 2 × 104 cells/well and cultured overnight before transfection. Following, cells were transfected with 0.7 μl of HiPerFect transfection reagent (Qiagen) mixed with 5 nm siRNA duplexes. After 24 h, incubation medium was replaced by starvation medium. 48 h later, BrdUrd labeling solution (Roche Applied Science) was added to the medium for 4 h, and the rate of proliferating cells was then determined as described above using the cell proliferation enzyme-linked immunosorbent assay (Roche Applied Science). The following siRNA duplexes were purchased from Qiagen: rat GSK3β, sense (r(CGAUUACACGUCUAGUAUA)dTdT) and antisense (r(UAUACUAGACGUGUAAUCG)dGdT); rat GSK3α, sense (r(GGGUGUAAAUAGAUUGUUA)dTdT) and antisense (r(UAACAAUCUAUUUACACCC)dAdA); control nonsilencing siRNA, sense (UUCUCCGAACGUGUCACGUdTdT) and antisense (ACGUGACACGUUCGGAGAA(dTdT)). Quantitative Reverse Transcription-PCR—Total RNA from 8 × 104 INS-1E cells growing on 4-cm2 surface area of a tissue culture dish was extracted using Qiagen RNAeasy kit according to the instructions of the manufacturer (Qiagen), and 2 μg was converted into cDNA. Primers for the analyzed genes were designed using the Primer Express 1.5 Software from Applied Biosystems, and sequences are available upon request. Quantitative real time PCR was performed using Applied Biosystems SDS 7000 detection system. Amplifications for a gene were performed in duplicate, and mean values were normalized to the mean value of the reference RNA, 18 S RNA. Mean gene expression values in cycle thresholds ± S.D. were determined from three wells of untreated rat islets kept for 24 h in culture medium (see Fig. 4E). The values for the indicated genes are as follows: bcl-xL, 23.6 ± 0.3; c-myc, 26.6 ± 0.4; id2, 22.7 ± 0.3; pax4, 32.2 ± 0.28; CDK4, 22.3 ± 0.2; neuroD, 19.35 ± 0.3; pdx1, 23.3 ± 0.2; cypB, 22.7 ± 0.3. A corresponding analysis was carried out with untreated INS-1E (see Fig. 3A). Mean cycle thresholds ± S.D. for GSK3α or -β and cyclophilin B were 21.5 ± 0.09, 25 ± 0.12, and 21.5 ± 0.1, respectively. GSK3 Inhibitors—The Chiron inhibitor CT99021 (6-{2-[4-(2,4-dichlorophenyl)-5-(4-methyl-1H-imidazol-2-yl)-pyrimidin-2-ylamino]ethyl-amino}nicotinonitrile) was synthesized by Asinex Inc. for DeveloGen AG. The compound was >98% pure by NMR and analytical high pressure liquid chromatography. All other GSK3 inhibitors used in this study were obtained from Calbiochem. Small Molecule Inhibitors of GSK3 Alleviate Glucolipotoxicity in INS-1E Beta Cells—INS-1E, a rat insulinoma cell line (42Merglen A. Theander S. Rubi B. Chaffard G. Wollheim C.B. Maechler P. Endocrinology. 2004; 145: 667-678Crossref PubMed Scopus (471) Google Scholar), shares many features with primary beta cells and is therefore widely used in the field to study beta cell function. We have studied the effects of well known and specific small molecule inhibitors of GSK3 on INS-1E cells exposed to toxic concentrations of glucose and the unsaturated fatty acid palmitate. INS-1E cells were treated for 24 h with a combination of high glucose (25 mm) and high palmitate (0.3 mm) concentrations. High glucose/high palmitate treatment was shown previously to induce cell death in INS-1-derived cell lines (44El-Assaad W. Buteau J. Peyot M.L. Nolan C. Roduit R. Hardy S. Joly E. Dbaibo G. Rosenberg L. Prentki M. Endocrinology. 2003; 144: 4154-4163Crossref PubMed Scopus (472) Google Scholar, 45Wente W. Efanov A.M. Brenner M. Kharitonenkov A. Koster A. Sandusky G.E. Sewing S. Treinies I. Zitzer H. Gromada J. Diabetes. 2006; 55: 2470-2478Crossref PubMed Scopus (412) Google Scholar) and induces endoplasmic reticulum stress in INS-1 cells (32Srinivasan S. Ohsugi M. Liu Z. Fatrai S. Bernal-Mizrachi E. Permutt M.A. Diabetes. 2005; 54: 968-975Crossref PubMed Scopus (152) Google Scholar, 46Wang H. Kouri G. Wollheim C.B. J. Cell Sci. 2005; 118: 3905-3915Crossref PubMed Scopus (231) Google Scholar). In fact, microscopic examination of INS-1E cells exposed to the mixture revealed typical features of apoptotic cells, such as condensed nuclei and membrane blebbing (data not shown). In order to quantitatively evaluate these apoptotic processes in the presence or absence of GSK3 inhibitors, two biological assays were established, enabling us to determine DNA fragmentation in the cytoplasm (Fig. 1, B, E, and H) and the enzymatic activity of caspases in cell lysates (Fig. 1, C, F, and I). Low levels of these apoptotic processes were detectable in untreated INS-1E cells (Fig. 1, Co.). The treatment with a combination of high glucose and palmitate stimulated these processes severalfold, confirming microscopic observations. For our studies, three structurally diverse, small molecule kinase inhibitors were selected: CHIR99021 (47Cline G.W. Johnson K. Regittnig W. Perret P. Tozzo E. Xiao L. Damico C. Shulman G.I. Diabetes. 2002; 51: 2903-2910Crossref PubMed Scopus (203) Google Scholar), 6-bromoindirubin-3′-oxime (BIO) (48Meijer L. Skaltsounis A.L. Magiatis P. Polychronopoulos P. Knockaert M. Leost M. Ryan X.P. Vonica C.A. Brivanlou A. Dajani R. Crovace C. Tarricone C. 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Treatment of INS-1E cells with 1-azakenpaullone or CHIR99021 alone did not compromise the viability of INS-1E cells even at high compound concentrations (Fig. 1, A and D, respectively). On the contrary, the GSK3 inhibitor BIO significantly reduced INS-1E viability at concentrations above 2 μm (Fig. 1G). In order to avoid potential off target effects, BIO was therefore used in the following studies at concentrations below 2 μm. Interestingly, all three GSK3 inhibitors counteract cell death induced by high glucose and high palmitate in INS-E cells in a concentration-dependent manner as demonstrated by reduced DNA fragmentation (Fig. 1, B, E, and H) and caspase activity (Fig. 1, C, F, and I), indicating a GSK3-mediated mechanism. On the other hand, GSK3 inhibitors did not completely abolish apoptotic processes, pointing at the involvement of GSK3-independent signaling events. Taken together, these results are consistent with previous reports demonstrating that inactivation of GSK3β protects beta cells against endoplasmic reticulum stress-induced apoptosis (32Srinivasan S. Ohsugi M. Liu Z. Fatrai S. Bernal-Mizrachi E. Permutt M.A. Diabetes. 2005; 54: 968-975Crossref PubMed Scopus (152) Google Scholar, 46Wang H. Kouri G. Wollheim C.B. J. Cell Sci. 2005; 118: 3905-3915Crossref PubMed Scopus (231) Google Scholar). Inhibition of GSK3 Activity Stimulates Growth of INS-1E Beta Cells—As previously reported by others, a functional PI 3-kinase/AKT pathway is instrumental for the regulation of beta cell mass. To study the role of the AKT target GSK3 in beta cell replication, INS-1E cells were incubated with the above described potent and specific GSK3 inhibitors. The treatment with any of the three GSK3 inhibitors increased the rate of proliferation of INS-1E cells in a dose-dependent manner as assessed by BrdUrd incorporation (Fig. 2, A, C, and E). The rate of proliferation reached a maximum in the presence of 2.5–10 μm CHIR99021 and 1-azakenpaullone, whereas BIO maximally stimulated proliferation between 0.5 and 1 μm. Above 1 μm, the proproliferative effects of BIO gradually disappeared, probably due to the off target effects already observed in Fig. 1G. GSK3 inhibitors promote survival and replication of INS-1E cells, implying that GSK3 inhibitor treatment may support net growth of INS-1E cells. In fact, INS-1E cell cultures treated with GSK3 inhibitors grew faster than vehicle-treated INS-E cell cultures, since cultures subjected for 4 days to GSK3 inhibitors contained more cells (Fig. 2, B, D, and F). Thus, GSK3 inhibitors appear to mimic growth factors and may be capable of stimulating the expansion of beta cell mass in vivo. In the course of the study, we also investigated the effects of other known small molecule GSK3 inhibitors on beta cell function (Table 1). 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