Cell Biology International (2006) 30, 1013-1017 - Robert Hägerkvist and others - Imatinib mesylate (Gleevec) protects against streptozotocin-induced diabetes and islet cell death in vitro

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Cell Biology International (2006) 30, 1013–1017 (Printed in Great Britain)

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Imatinib mesylate (Gleevec) protects against streptozotocin-induced diabetes and islet cell death in vitro

Robert Hägerkvist^a, Natalia Makeeva^a, Stephen Elliman^b and Nils Welsh^a^*

^aDepartment of Medical Cell Biology, Uppsala University, Biomedicum, P.O. Box 571, Husargatan 3, S-75123 Uppsala, Sweden
^bDiabetes and Metabolism Disease Area, Novartis Institutes for BioMedical Research, Cambridge, MA 02139, USA

Abstract

The tyrosine kinase inhibitor imatinib mesylate (Gleevec) has been demonstrated to protect various cell types from death by inhibition of Abelson tyrosine kinase (c-Abl). The aim of the present study was to establish whether imatinib protects the insulin producing β-cell from the different apoptosis promoting agents in vitro and whether imatinib counteracts streptozotocin-induced diabetes in NMRI mice. We observe that imatinib attenuated the actions of several different death promoting substances. In addition, mice injected with streptozotocin did not develop diabetes when given imatinib. The beneficial effects of imatinib may be related to inhibition of the pro-apoptotic MAP kinase JNK. We conclude that imatinib protects against β-cell death and that this may contribute to the previously reported anti-diabetic actions of imatinib.

Keywords: Imatinib, Pancreatic islet, Diabetes, JNK, Apoptosis.

^*Corresponding author. Tel.: +46 18 42 12; fax: +46 18 471 40 59.

1 Introduction

Imatinib mesylate, also known as Gleevec or Glivec, is a selective tyrosine kinase inhibitor that specifically inhibits cellular Abelson tyrosine kinase (c-Abl), platelet derived growth factor receptor (PDGFR), transmembrane receptor tyrosine kinase (c-Kit), and Abl related gene (Arg) (Buchdunger et al., 1996; Okuda et al., 2001). Imatinib is successfully used in the clinic to treat malignancies such as chronic myeloid leukemia and gastrointestinal stromal tumors (O'Brien et al., 2003; Demetri et al., 2002). Furthermore, it has recently been observed that a modest number of patients, suffering from both chronic myeloid leukemia and Type 2 diabetes, were successfully treated for not only their leukemia, but also for diabetes, when given imatinib (Veneri et al., 2005; Breccia et al., 2004). The molecular mechanisms underlying the beneficial effects of imatinib in these cases are unknown, but may be related to the propensity of imatinib to inhibit non-receptor tyrosine kinase c-Abl. Indeed, many studies have shown that imatinib, by blocking c-Abl phosphorylation activity, can prevent the death of various cell types (Kumar et al., 2003; Raina et al., 2002, 2005).

Type 1 diabetes is an autoimmune disease in which dysfunction and damage of insulin-producing β-cells is thought to arise from direct contact with immune cells and from exposure to cytotoxic pro-inflammatory cytokines and nitric oxide (Eizirik and Mandrup-Poulsen, 2001). In Type 2 diabetes β-cells are also dysfunctional and damaged, possibly in response to peripheral insulin resistance, hyperglycemia, hyperlipidemia and cytokines, leading to a relative lack of insulin (Cnop et al., 2005). The molecular events leading to cytokine-induced β-cell dysfunction and death have been investigated and it appears that the activation of mitogen-activated protein kinases (MAPK), such as JNK, ERK and p38, in response to both cytokines (Eizirik and Mandrup-Poulsen, 2001) and nitric oxide (Welsh, 1996), plays a central role in this chain of events.

To our knowledge it has hitherto not been studied whether imatinib has a protective effect on the insulin producing β-cell. Therefore, the aim of the present investigation was to determine whether imatinib affects β-cell death in vitro and in vivo. In order to kill β-cells and to induce diabetes in vivo, streptozotocin (STZ) is commonly used, which causes a rapid and selective destruction of β-cells with resulting hyperglycemia when injected in vivo (Rakieten et al., 1963). This model relies solely on intrinsic β-cell destruction, without the involvement of peripheral insulin sensitivity or the immune system. We report here that β-cell sensitivity, not only to STZ, but also to other apoptosis-promoting agents, including pro-inflammatory cytokines, is decreased by imatinib and that this protection could involve the decreased activation of the pro-apoptotic MAP kinase JNK.

2 Materials and methods

2.1 Animals, islet isolation and tissue culture

Male NMRI (Naval Medical Research Institute-established, Mölle och Bomholt gård, Denmark) and Sprague–Dawley rats (local colony at Biomedical Center, Uppsala, Sweden) were kept under standard pathogen free conditions, with free access to tap water and pelleted food. Islets were isolated by a collagenase digestion procedure and precultured as previously described (Sandler et al., 1987). Local animal ethics committee at Uppsala University approved all experiments. C2C12 mouse skeletal myoblasts (ATCC #CRL-1772) were cultured in DMEM High Glucose supplemented with 10% FBS (Growth Medium). To differentiate C2C12 myoblasts into myotubes, cells were permitted to reach 80% confluence and the serum concentration was reduced to 2% horse serum.

2.2 STZ treatment in vivo

Male NMRI mice were divided in groups and injected with 0.2ml freshly dissolved STZ (Sigma) (120mg/kg bodyweight) in 0.9% NaCl, or saline alone into the tail vein. The mice received either gavage with 0.9% NaCl or imatinib (Novartis, Basel, Switzerland) (200mg/kg bodyweight) the day before, 2h before the intravenous STZ/saline injection and the day after the injection. Blood glucose levels were determined by blood samples from the tail tip using ExacTech blood glucose meter (Baxter Travenol, Deerfield, IL, USA).

2.3 Islet treatment in vitro

Groups of islets were pre-treated with imatinib (10μM) for 2 or 18h, followed by exposure to Staurosporine (5μM), Thapsigargin (200nM), hydrogen peroxide (100μM), FCCP (p-trifluoromethoxy carbonyl cyanide phenyl hydrazone) (5μM), STZ (0.75mM in glucose free medium) or a combination of cytokines (IL-1β (25U/ml)+IFN-γ (1000U/ml)+TNF-α (1000U/ml)) for the indicated time points.

2.4 Evaluation of islet viability

Islets were vital stained with propidium iodide (Sigma) (20μg/ml) and bisbenzimide (Sigma) (5μg/ml) for 15min at 37°C. After careful washing, islets were placed on coverslips and examined by fluorescence microscopy using Openlab 3.0.4 software. The total number of cells as well as necrotic and apoptotic nuclei were counted with NIH Image 1.63 software.

2.5 Treatment and harvest of myotubes in vitro

72h post-differentiation cells were treated with hydrogen peroxide and harvested as follows: growth media was removed and cells were washed with PBS. Cells were trypsinised to remove myotubes and leave myoblasts adhered to the plate. Myotubes were resuspended in growth media to inactivate the trypsin and washed once in PBS. Cell were lysed in RIPA buffer (50mM Tris, pH 7.4, 1% NP-40, 0.25% Na-deoxycholate, 150mM NaCl, 1mM EDTA, 1mM Na3VO4, 1mM NaF) and supplemented with one Protease inhibitor tablet (Roche). Lysates were centrifuged at 10,000rpm at 4°C, supernatants harvested and equal quantities of protein were used for immunoblotting as described below.

2.6 Immunoblotting

Islets were washed with ice cold PBS and directly suspended in SDS-sample buffer (2% SDS, 0.15M Tris pH 8,8, 10% glycerol, 5% β-mercaptoethanol and bromophenolblue), supplemented with 2mM PMSF, boiled for 5min and separated on SDS-PAGE. Proteins were electrophoretically transferred to Immobilon filters. Filters were blocked in 2.5% milk powder for 1h, after which they were probed with anti-phospho JNK, c-Jun, ERK and p38 antibodies (Cell Signaling Technology, Danvers, MA, USA), then with anti-total-JNK, c-Jun, ERK or p38 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After a brief wash with PBS +0.1% Tween 20, membranes were probed with HRP-linked secondary antibodies (1:1000) for 1h and extensively washed. The bound antibodies were visualized using ECL (Amersham) and were quantified by densitometry using Kodak image analysis software version 3.6.

3 Results and discussion

Imatinib is known to protect against genotoxic agent-, death receptor activation- and hydrogen peroxide-induced apoptosis in various cell types via inhibition of the c-Abl kinase (Raina et al., 2005; Dan et al., 1999; Kumar et al., 2003). To test whether imatinib also protects insulin producing cells, we pre-treated rat islets with 10μM imatinib and exposed them to various inducers of cell death; staurosporine (general kinase inhibitor), thapsigargin (endo/sarcoplasmic calcium ATPase inhibitor), hydrogen peroxide (oxidative stress), FCCP (mitochondrial uncoupling) and STZ (DNA alkylation, oxidative stress, depletion of NAD). As depicted in Fig. 1A these various agents all augmented islet cell death. We also observed that imatinib, at least partially, protected the islet cells from death induced by the different toxins (Fig. 1A). This indicates that imatinib promotes survival by inhibiting a signaling event common for several pathways leading to β-cell death. Assuming that the activity of c-Abl is linked to death signaling in response to events such as ER stress, oxidative stress or a sustained elevation of the cytosolic Ca²⁺ concentration, it might be possible to block a multitude of death signals that participate in the pathogenesis of diabetes employing only one approach.

Fig. 1

A. Imatinib protects against a variety of islet cell death inducing agents in vitro. Rat islets in groups of 10 were pre-incubated with 10μM of imatinib for 2h. Islets were then cultured with hydrogen peroxide (H2O2, 100μM), FCCP (5μM) and STZ (0.75mM) for 20h or for 72h with staurosporine (Stau, 5μM) and thapsigargin (Thap, 200nM). This was followed by vital staining and the total number of cells as well as necrotic and apoptotic nuclei were counted with NIH Image 1.63. Results are means±SEM for two separate observations. B. Imatinib protects against pro-inflammatory cytokines in vitro. Mouse pancreatic islets were incubated with imatinib (10μM) overnight. This was followed by exposure to a mix of cytokines for 24h. Cell death was quantified by vital staining and fluorescence microscopy. Results are means±SEM for 3–4 observations. * denotes p<0.05 using Student's t-test.

Destruction of insulin producing cells in Type 1 diabetes may in part be mediated by the pro-inflammatory cytokines IL-1β, IFN-γ and TNF-α (Eizirik and Mandrup-Poulsen, 2001). Therefore, after pre-treatment with 10μM imatinib overnight, we exposed mouse islets in vitro to this combination of cytokines for 24h, followed by vital staining and evaluation of islet cell viability. Interestingly, we observed that treatment with imatinib potently prevented islet cell death induced by pro-inflammatory cytokines (Fig. 1B).

Having observed that imatinib prevented β-cell death in vitro, we next gavaged male NMRI mice with saline alone or 200mg imatinib/kg bodyweight, the day before, 2h before an intravenous STZ injection and the day after the STZ injection. As shown in Fig. 2 the imatinib treatment protected completely against hyperglycemia induced by the STZ injection. This finding suggests that imatinib prevented STZ induced β-cell apoptosis/necrosis in vivo.

Fig. 2

Imatinib protects NMRI mice from STZ-induced hyperglycemia in vivo. The mice were either injected/gavaged with saline (n=5 open square), gavaged with imatinib (n=5 black rhomb), injected with STZ (n=10 black square) or both gavaged with imatinib and injected with STZ (n=10 open rhomb). The imatinib/saline gavage was given on day −1, 0 and 1. On day 0 the mice were injected with 120mg/kg STZ intravenously 2h after the administration of imatinib and the blood glucose was determined on the days given in the figure. Points are means±SEM. *denotes p<0.05 vs. corresponding STZ+imatinib group using Student's t-test.

The activation of iNOS and ensuing NO formation in response to cytokine stimulation is known to be a major contributor of cytokine-induced β-cell death in rodents (Eizirik and Mandrup-Poulsen, 2001) and addition of either cytokines or NO-donors to islets cultured in vitro has been reported to activate pro-apoptotic MAP kinases JNK and p38 (Welsh, 1996; Makeeva et al., 2006). In addition, in cell types other than β-cells, activation of cytosolic c-Abl has been shown to result in phosphorylation of MAP and ERK Kinase-1 (MEKK-1), which in turn promotes MKK4 and JNK1/2 activation (Kharbanda et al., 2000). We therefore investigated the possibility that imatinib affected MAPK activity. For this purpose, rat islets were incubated for 24h with 10μM imatinib and subsequently exposed to the NO-donor DETA/NO (2mM) for 20min. Islets were then analyzed for phosphorylation of p38, JNK2 and ERK1/2 using phosphospecific antibodies and immunoblotting. We observed that imatinib significantly decreased JNK2 phosphorylation in response to DETA/NO (Fig. 3A).

Fig. 3

Imatinib decreases NO-induced JNK activation in rat pancreatic islets. Rat islets were pre-incubated for 24h with 10μM imatinib and then exposed to DETA/NO (2mM) for 20min. Relative activation of p38, JNK and ERK levels was quantified by relating phospho-protein bands to total-protein bands. Bars are means±SEM for 4 independent observations. *denotes p<0.05 vs corresponding non-imatinib treated islets using ANOVA and Student's paired t-test.

There was also a trend towards a lowered DETA/NO-induced activation of p38 and ERK1/2 as a result of the imatinib-treatment, but it did not reach statistical significance (Figs. 3B, C). The trend to a lowered p38 and ERK activation is in line with the established role of imatinib to act at a site upstream of MAP kinases, probably with c-Abl, rather than directly with JNK, ERK or p38. To determine whether imatinib affected JNK phosphorylation in cells other than islet cells, we also investigated hydrogen peroxide-induced JNK phosphorylation in myotubules. In good agreement with the effects observed in islets, JNK phosphorylation in myotubules was also diminished by imatinib (Fig. 4). This was further supported by the finding that phosphorylation of the JNK substrate c-Jun was also decreased in response to imatinib (Fig. 4). Thus, imatinib-mediated inhibition of JNK could be one mechanism by which imatinib prevents β-cell death. This is in line with a previous report stating that cell-permeable inhibitors of JNK protect against cytokine-induced cell death in insulin producing cells (Bonny et al., 2001).

Fig. 4

Imatinib decreases hydrogen peroxide-induced JNK activation in murine C2C12 myotubes. Postmitotic murine C2C12 myotubes were pre-incubated for 2h with either DMSO or 20μM imatinib and then challenged with increasing doses of hydrogen peroxide (100–500μM) for 1h. Results are representative of 3 experiments.

In summary, our findings indicate that β-cells are protected from death both in vitro and in vivo by imatinib, and that this protection involves a lowering of the JNK activity. Hypothetically, this could explain, at least in part, the beneficial effects observed by imatinib in Type 2 diabetes (Veneri et al., 2005; Breccia et al., 2004). Unfortunately, due to the side effects that have been observed in the clinic, it is not likely that imatinib will be used as a treatment for diabetes. Instead, the only curative treatment for Type 1 diabetes is islet transplantation combined with immunosuppressive therapy (Shapiro et al., 2005). However, it has been reported that up to 60% of the transplanted islet mass is rapidly lost due to apoptosis, in part dependent on hypoxia-induced damage (Emamaullee et al., 2005). The apoptosis rate in the graft reaches its peak 2–3days after transplantation, then it declines until day 14 (Emamaullee et al., 2005). Therefore, studies that could establish whether pre-treatment with imatinib, either ex vivo or in vivo, could enhance graft survival and function during the first two critical weeks post-transplantation are highly warranted.

Acknowledgements

This work was supported in part by the Swedish Research Council (12X-11564), the Swedish Diabetes Association, the family Ernfors Fund, the Novo-Nordisk Fund and the European Foundation for the Study of Diabetes.

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Received 22 May 2006/14 July 2006; accepted 6 August 2006

doi:10.1016/j.cellbi.2006.08.006

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