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1 Division of Endocrinology, Cedars-Sinai Medical Center, Los Angeles, California, USA
2 Universita’ ‘Magna Graecia’, Catanzaro, Italy
3 University of California, Los Angeles, California, USA
4 Department of Clinical Sciences, University ‘La Sapienza’, Rome, Italy
(Requests for offprints should be addressed to R Perfetti; Email: perfettir{at}cshs.org)
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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A recent review of the potential barriers to insulin independence after islet transplantation identified several factors (Hering & Ricordi 1999). The number of ß-cells may be inadequate owing to insufficient engraftment of islets and immediate cellular loss through apoptosis and other non-immune-mediated inflammatory pathways (Bennet et al. 1999). A persistent impairment of glucose metabolism after transplantation has been attributed to various causes, including impaired insulin secretion (Patty et al. 2002) and ß-cells apoptosis induced by the administration of immunosuppressive drugs (ISD) (Drachenberg et al. 1999).
For any type of immunossuppression after transplantation, a balance is sought between efficacy and toxicity. A major advance toward achieving insulin independence following islet transplantation or pancreas transplantation is to eliminate, or minimize, the use of immunosuppressive agents known to have ß-cell toxicity (Zeng et al. 1993) or to induce peripheral insulin resistance (Sutherland et al. 2001). An alternative strategy might consider ways to enhance the resistance of islets to the toxic effects of ISD.
Recent reports have shown that the gastrointestinal hormone glucagon-like peptide 1 (GLP-1), in addition to regulating the secretion of insulin and the expression of islet-specific genes (Hussain & Habener 2000, Drucker 2003), has significant antiapoptotic properties. In diabetic ZDF rats, as well as in streptozotocin-induced diabetic mice, treatment with GLP-1 or exendin-4 (a long-acting analog of GLP-1) protects ß-cells from apoptosis (Farilla et al. 2002, Li et al. 2003). In vitro studies using insulin-secreting cells (Hui et al. 2002) or fibroblast-expressing GLP-1 receptors (Li et al. 2003) have shown that the protective action of GLP-1 is not an indirect effect resulting from the normalization of the ambient glucose levels, but rather it has a direct effect on cell viability and is mediated by an increase inexpression of antiapoptotic proteins and down-regulation of proapoptotic proteins (Farilla et al. 2002, Hui et al. 2003, Li et al. 2003).
The aim of this study was to investigate whether GLP-1 would confer resistance to the toxic effects of ISD, preserving the viability and function of ß-cells. Using our previously described ß-cell line transfected with the GLP-1 fragment of the human proglucagon gene (Hui et al. 2002), we demonstrate that GLP-1 counteracts the negative effects of ISD, improving the glucose-dependent capability to secrete insulin and reducing ß-cell death.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Mouse insulinoma (MIN-6) cells were a gift from Dr Junichi Miyazaki (Kumamoto University; Kumamoto, Japan). Transfection of MIN6 cells with the GLP-1 fragment of the human proglucagon gene, under the control of the rat insulin II promoter (RIP/GLP-1 construct) has been previously described by Hui et al.(2002). RIP/GLP-1 MIN6 cells have been shown to be capable of promoting the synthesis and secretion of GLP-1 and insulin in a glucose-dependent manner. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) medium (Life Technologies Inc., Gaithersburg, MD, USA) containing100 µg/ml penicillin, 50 µg/ml streptomycin, and 10% fetal bovine serum (FBS) (Gemini Bio-Products Inc., Woodland, CA, USA) at 37 °C under a humidified condition of 95% air and 5% CO2. All cell culture studies were conducted using cells grown to 80% confluence and exposed for 48 h to medium containing a cocktail of ISD, or an equivalent volume of the medium used to dilute each of the drugs used. The ISD used included sirolimus (Rapamycin, Sigma Aldrich, St Louis, MO, USA) at concentrations of 25 ng/ml; mycophenolate, (Mycophenolic acid, Calbiochem, La Jolla, CA, USA) at concentrations of 17.5 ug/ml; and tacrolimus (Prograf, Fujisawa, Deerfield, NJ, USA) at concentrations of 75 ng/ml. Drug dilutions were made from fresh stock before each experiment. Sirolimus was solubilized in dimethyl sulfoxide, mycophenolate in 70% ethanol, and tacrolimus in sterile water. The selection of those agents was based on the protocol for immunosuppression often employed after pancreatic islets transplantation (Shapiro et al. 2000), and the concentrations of ISD used in the present study were selected on the basis of previous experiments performed in our laboratory (Hui et al. 2004).
In some studies, human recombinant GLP-1 (American Peptide Company; Sunnyvale, CA, USA) was added to the culture medium of parental MIN6 cells. Those experiments were aimed at investigating whether the effect of ISD on RIP/GLP-1 MIN6 cells was specifically due to their capability of producing GLP-1, or whether it was the effect of a clonal selection resulting from the process of cell transfection. Cells were evaluated for insulin secretion, viability, and the expression of anti-apoptotic and pro-apoptotic factors. The specific culture conditions for each individual experiment are detailed in the specific experimental protocol described hereafter. All experiments with transfected MIN6 cells were performed using pools of stably transfected clones.
Measurement of insulin secretion
Parental MIN6 and RIP/GLP-1 MIN-6 cells were plated at a density of 1 x 106 cells/well in 6-wells plates. After they reached confluence, cells were subjected to a 2 h wash-out and cultured in serum-free medium containing 25 mM glucose and a cocktail of ISD (sirolimus (25 ng/ml), mycophenolate (17.5 ug/ml) and tacrolimus (75 ng/ml)) for 48 h. Control cultures were incubated in the absence of ISD for the same length of time. After a 48 h treatment the cells were subjected to a 2 h wash-out period in serum-free/glucose-free medium, and exposed for 1 h to medium containing either 5.6 or 16.7 mM glucose. The level of insulin in the culture medium was measured by RIA (Linco Research Inc., St Charles, MA, USA) and normalized for the total cellular protein content detected in the pellet of each individual culture, as measured using the Bradford method (Bio-Rad Laboratories Inc., Richmond, CA, USA).
LIVE/DEAD viability/cytotoxicity
Parental and RIP/GLP-1 MIN6 cells were grown in chamber slides and treated with a cocktail of ISD and analyszed with the LIVE/DEAD Viability/Cytotoxicity Kit (Probes, Eugene, OR, USA). After 48 h treatment, the cells were collected, washed twice with PBS, stained for 30 min, and visualized under UV light with a BH-2 microscope (Olympus Corp., Melville, NY, USA). This is a fluorescence-based method for determining viability of adherent or non-adherent cells and for assaying cytotoxicity. The assay comprises of two probes: calcein AM and ethidium homodimer-1. Calcein AM is a fluorogenic esterase substrate that is hydrolyzed to a green-fluorescent product (calcein); thus, the emission of green fluorescent light is an indicator of cells that have esterase activity as well as an intact membrane to retain the esterase products. Ethidium homodimer-1 is a high-affinity, red-fluorescent nucleic acid stain that is only able to pass through the compromised membranes of dead cells.
Immunostaining for insulin and the active form of caspase-3
After a 48 h treatment with or without ISD, cells were fixed for 1 h at room temperature in 4% paraformaldehyde in PBS (pH 7.4). They were then washed in PBS, permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, rinsed twice in PBS, and incubated with a polyclonal goat anti active form of caspase-3 antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:250 in PBS, or with a polyclonal guinea pig anti-insulin antibody (DAKO Corp., Carpinteria, CA, USA) diluted 1:400. The incubation was performed at 4 °C overnight. After several washes in PBS, an antigoat fluorescein-conjugated antibody (Probes, Eugene, OR, USA) diluted 1:1500 for the detection of caspase-3, and an anti-guinea pig rhodamine-conjugated antibody (Probes), diluted 1:1500 for the detection of insulin, were incubated for 1 h at room temperature. To identify the nuclei, the cells were also stained with the karyophilic dye Hoechst 33342 (10 µ g/ml) for 5 min at room temperature. After a final wash in PBS, the cells were visualized under UV light with a BH-2 microscope (Olympus Corp., Melville, NY, USA) and using a fluorescein standard filter set (520 ± 20 nm) for caspase-3 staining and a rhodamine filter (>620 nm) for insulin.
FACS analysis for Annexin-V
Apoptotic cells were analyzed by flow cytometer (FACS; Becton Dickinson and Co., San Jose, CA, USA) using the Annexin-V-FLOUS Staining Kit (Roche Diagnostic Corporation, Indianapolis, IN, USA). Annexin-V is a Ca2+-dependent phospholipid-binding protein with a high affinity for phosphotidylserine (PS); hence, this protein can be used as a probe for PS exposure on the outer leaflet of the cell membrane and can be used for the detection of apoptotic cells. The simultaneous application of propidium iodide as a DNA stain, used for dye exclusion tests, allows Annexin-V positively stained cells to be distinguished from necrotic cells. Cells were collected from the culture flasks and washed twice with PBS by centrifugation at 200 g for 5 min. The cell pellet was then suspended in 100 µl of staining solution and incubated for 15 min at room temperature. Flow cytometric analysis was performed with a FACScan cytometer (Becton Dickinson and Co., Franklin Lakes, NJ, USA), using the LYSIS II analyzer program. Exendin (9–39) was purchased from American Peptide Co. (Sunnyvale, CA, USA).
DNA fragmentation analysis
Floating and adherent cells from each culture condition studied were collected, centrifuged, pelleted at 400 g for 5 min, and washed twice with PBS. The pellet was then resuspended in 0.2 ml lysis buffer (100 mM NaCl, 10 mM Tris (pH 8.0), 1 mM EDTA, 0.5% sodium dodecyl sulfate, 0.20 mg/ml proteinase K, 200 µg/ml ribonuclease A). The cell lysates were incubated at 37 °C for 2 h. The genomic DNA was extracted by two separations with phenol/chloroform, followed by one extraction with chloroform alone. The DNA pellet was then washed in 70% ethanol and resuspended in 1 mM EDTA and 10 mM Tris–HCl (pH 8.0) at a final concentration of 20 µg/ml. The DNA fragmentation analysis was performed using a 1.5% agarose gel in 1 mM EDTA, 40 mM Tris acetate (pH 7.6) to visualize the laddering of the samples.
Western blot analysis
Cell pellets were lysed at 4 °C in a buffer containing 60 mM Tris–HCl (pH 6.8), 1% sodium dodecyl sulfate, 10% glycerol, 0.05% NP-40 and 0.5% ß-mercaptoethanol and protease inhibitor mixture (1:100 dilution). The lysis buffer for the protein extraction to determine the cytoplasmic abundance of Smac was prepared with the same reagents listed above, without NP-40. Lysates were cleared at 12 000 r.p.m. for 15 min at 4 °C and stored at –80 °C until used. The protein concentration was determined using a Comassie dye assay (Bio Rad, Hercules, CA, USA), and BSA (Sigma Aldrich) was used as a standard. The cell lysates (25 µg per sample) were then separated by 8% or 12% SDS-PAGE under reducing conditions and electrotransferred onto Hybond-C nitrocellulose membrane (Bio Rad) using standard procedures. The membranes were incubated for 2–4 h at room temperature with TBST (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 0.2% Tween-20) detection reagent. Primary antibodies were used at the following dilutions: anti Bcl-2 (1:500); anti poly-(ADP-ribose)-polymerase (PARP; 1:500); anti second mitochondria-derived activator of caspase Smac/DIABLO (1:400); anti active form of caspase-3 (1:1000); and anti ß-actin (1:1000). All antibodies were purchased from Santa Cruz Biotechnology Inc.
Measurement of GLP-1 secretion
Parental and RIP/GLP-1 MIN6 cells utilized to perform FACS analysis for Annexin-V were also used to evaluate the concentration of GLP-1 in their culture medium. GLP-1 was measured by RIA (Linco Research Inc., St Charles, MA, USA).
Statistical analysis
The data were expressed as mean ± S.E. Comparisons of means of multiple groups were made using one-way ANOVA followed by post hoc analysis using Fisher’s least significant difference method.
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Results |
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The exposure of parental MIN6 cells to a cocktail of ISD for 48 h produced a significant reduction of their ability to secrete insulin after glucose challenge. Indeed, when parental MIN6 cells were exposed (for 1 h) to medium containing 16.7 mM glucose (after a 2 h wash out with glucose-free, serum-free medium) there was a significantly decrease in the insulin concentration in cultures exposed to ISD (1.02 ± 0.21 ng insulin/mg pellet protein) compared with cultures exposed to vehicle alone (1.71 ± 0.19 ng insulin/mg pellet protein; P<0.01) (Fig. 1
). Conversely, RIP/GLP-1 MIN6 cells showed a significant preservation of glucose-dependent secretion of insulin after treatment with ISD. Indeed, exposure of RIP/GLP-1 MIN6 cells to ISD was associated with an accumulation of insulin in the culture medium that was equivalent to approximately 85% of the total insulin accumulated in the medium of untreated RIP/GLP-1 MIN6 cells (Fig. 1).
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Qualitative analysis of the effect of GLP-1 on cell viability
The viability of parental and RIP/GLP-1 MIN6 cells exposed to either a cocktail of ISD or vehicle for 48 h was measured using calcein-AM (a membrane-permeable dye that in live cells is cleaved by esterases to yield cytoplasmic green fluorescence) and ethidium homodimer-1 (a red nucleic acid dye to which live cells are impermeable, but that accumulates in the nuclei of membrane-compromised cells) staining. Panel A and B of Figure 2 show that in cultures of both parental MIN6 cells and RIP/GLP-1 MIN6 cells grown without ISD virtually all cells were viable. Both cultures showed a large number of dead cells after exposure to ISD. However, cultures of parental MIN6 cells (C) showed a greater number of dead cells, compared with cultures of RIP/GLP-1 MIN6 cells (D).
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). This experiment indicated that 18.83 ± 5% of parental MIN6 cells vs. 8.33 ± 5% of the RIP/GLP-1 MIN6 cells were Annexin-V-positive when exposed to ISD (P<0.01). The transfection of cells with GLP-1 was also associated with significant protection from necrosis induced by ISD (P<0.01) and an overall larger percentage of living cells (75.64 ± 5% vs. 58.51 ± 5% in RIP/GLP-1 MIN6 cells vs. parental MIN6, P<0.01), after challenge with ISD (P<0.01; Table 1).
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GLP-1 inhibited the DNA fragmentation induced by ISD
Activation of a calcium-dependent endonuclease is one of the earliest detectable events denoting an irreversible commitment to cell death. This results in the characteristic formation of DNA fragments of 180–200 bp. Electrophoresis of genomic DNA from MIN6 cells exposed to ISD showed the characteristic laddering pattern that leads to cell death (Fig. 3). This was in clear contrast with the preserved DNA integrity that was observed in RIP/GLP-1 MIN6 cells exposed to ISD.
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GLP-1 increased the intracellular levels of Bcl-2
RIP/GLP-1 MIN6 cells exhibited a significantly greater abundance of the anti-apoptotic protein Bcl-2 compared with parental cells (Fig. 4), and its abundance was minimally affected by the exposure of cells to ISD.
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). In comparison, RIP/GLP-1 MIN6 cells cultured in the presence of the ISD showed a significant preservation of the PARP in its inactive form, and exposure of RIP/GLP-1 MIN6 cells did not alter the intracellular level of p85.
GLP-1 prevents the mitochondrial release of Smac/DIABLO
Parental MIN6 exposed to the cocktail of ISD for 48 h exhibited a significant increase in Smac/DIABLO levels compared with untreated cultures (Fig. 4). This effect was significantly reduced in RIP/GLP-1 MIN6 cells, even after exposure to ISD.
GLP-1 inhibits the activation of caspase-3
The exposure of parental MIN6 cells to ISD produced a detectable activation of caspase-3, as determined by immunostaining (Fig. 5, top panel). Conversely, RIP/GLP-1 MIN6 cells were resistant to the activation of caspase-3 after exposure to ISD.
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, bottom panel), and demonstrated that while in parental MIN6 cells the exposure to ISD produced a 4.5-fold increase in the intracellular level of the active form of caspase-3 (ISD-treated cells vs. cells cultured with vehicle alone; P<0.001), the effect of ISD on RIP/GLP-1 MIN6 cells was less pronounced. Indeed, RIP/GLP-1 MIN 6 cells showed an induction in the activation of caspase-3 of 1.9 fold when compared with cells cultured with vehicle alone (P<0.01), and this was equivalent to approximately 60% lower than parental MIN6 cells cultured under the same conditions (ISD-treated MIN6 cells vs. ISD-treated RIP/GLP-1 MIN6 cells, P<0.001). The anti-apoptotic effect of GLP-1 was independent from its effect on insulin secretion
In order to investigate whether the capability of RIP/GLP-1 MIN6 cells to counteract the pro-apoptotic action of ISD was a direct effect of GLP-1, or whether it was the result of a greater secretion of insulin in cells expressing GLP-1, we cultured cells in the presence of diazoxide (250 µM/L for 48 h), an agent known to inhibit insulin secretion. The viability of cells was investigated using the DNA laddering assay and their capability to secrete insulin was measured using a RIA. We demonstrated that, while diazoxide was effective in suppressing the secretion of insulin (2.36 ± 0.21 ng of insulin/mg of protein in untreated cultures vs. 0.41 ± 0.07 ng of insulin/mg of protein; P<0.01), its action did not diminish the resistance of RIP/GLP-1 MIN6 cells to the pro-apoptotic action of ISD (Fig. 6, lanes D vs. E).
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, lane C). In contrast, RIP/GLP-1 MIN6 cells did not show DNA fragmentation when exposed to ISD. These results supported the hypothesis that the antiapoptotic role of GLP-1 on the cells exposed to a cocktail of ISD was independent from its effect on the stimulation of insulin secretion. Inhibition of GLP-1 receptor activation by exendin 9 abolished the effect of GLP-1 on the viability of cells exposed to ISD
To investigate whether the capability of RIP/GLP-1 MIN6 cells to counteract the proapoptotic effect of ISD was specifically mediated by the activation of the GLP-1 receptor, cells were cultured in medium containing increasing concentrations of the receptor antagonist exendin-9. FACS analysis for Annexin V demonstrated that 19.60 ± 2% of parental MIN6 cells vs. 8.34 ± 2.3% of RIP/GLP-1 MIN6 cells were Annexin-V-positive when cultured for 48 h in medium containing ISD (P<0.01) (Fig. 7). The cell survival advantage that RIP/GLP-1 MIN6 cells had when compared with parental MIN6 cells was abolished by the presence of exendin 9 in the culture medium. Indeed, as the concentration of exendin 9 in the medium was increased, the difference in the percentage of living cells between the two cell lines (RIP/GLP-1 MIN6 cells and parental MIN6 cells) became no longer significant. The capability of exendin-9 to inhibit cell death reached its plateau at the concentration of 10 nM, and at that concentration the percentage of death cells was very similar among cultures (43.4 ± 2.1 vs. 41.7 ± 1.9, in parental vs. RIP/GLP-1 MIN6 cells, respectively) (Fig. 7).
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GLP-1 induced a dose-dependent inhibition of cell death
To investigate the dose-dependent inhibition of cell death by GLP-1 we cultured parental MIN6 cells in the presence of various concentration of GLP-1 (for 48 h) and then exposed each culture condition to ISD (for an additional 48 h). In addition to defining the dose response for GLP-1's effect on cell viability, the aim of this experiment was to investigate whether the observed capability of RIP/GLP-1 MIN6 to be protected against the toxic effect of ISD was due to the specific action of GLP-1, or whether it was the result of a clonal selection that rendered the transfected cell line less susceptible to cell death. FACS analysis for Annexin-V demonstrated the presence of a dose-dependent inhibition of cell death by GLP-1 (Fig. 8). The presence of 1 nM GLP-1 in the culture medium was sufficient to produce a statistically significant inhibition of cell death (38% vs. 29.7% in control vs. GLP-1 treated cultures, respectively; P<0.01) and it further increased in a dose-dependent fashion in cultures grown in 5, 10 or 100 nM of GLP-1.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Our findings are consistent with previous reports describing deterioration in islet function and viability in response to tacrolimus, sirolimus and mycophenolate. Drachenberg et al.(1999) performed electron microscopy studies of pancreatic biopsies from patients subjected to pancreas and kidney transplantation and showed that treatment with tacrolimus was associated with an increased cytoplasmic swelling, cellular vacuolization, and a marked decrease in dense-core secretory granules of islet cells. Paty et al.(2002) demonstrated that mycophenolate and tacrolimus had deleterious effects on insulin secretion in both HITT15 cells and cultured rat islets. In addition to inhibiting insulin secretion (Ricordi et al. 1991, Strasser et al. 1992, Ishizuka et al. 1993), tacrolimus has been shown to down regulate the synthesis of insulin (Redmon et al. 1996). Mycophenolate has been shown to reduce insulin secretion by inhibiting voltage-dependent calcium channels (Meredith et al. 1997, Li et al. 2000). On the other hand, sirolimus has been shown to have a very modest adverse effect on islet function (Fabian et al. 1992, Kneteman et al. 1995); and a reduction in insulin release has been demonstrated only after a prolonged culture with a high concentration of the drug (Fabian et al. 1992). Prior observations from various laboratories have shown that GLP-1 was capable of inducing the expression of anti-apoptotic proteins leading to improved survival of cells. Li et al.(2003) have shown that GLP-1 receptor signaling modifies the susceptibility of mice to undergo islet cell apoptosis after treatment with streptozotocin. Farilla et al.(2002) have demonstrated that GLP-1 reduced the number of apoptotic cells in the pancreas of Zucker diabetic rats, and that GLP-1 when added to freshly isolated human islets or cultured MIN6 cells preserved cell morphology, enhanced insulin synthesis, and improved the cellular capability of secreting insulin in response to glucose (Farilla et al. 2003, Hui et al. 2003). Interestingly, a protective effect of GLP-1 from cellular apoptosis has also been shown on cells other than the pancreatic endocrine cells, as for the neuronal cells of hippocampus (During et al. 2003).
In the present report, studying factors that may mediate the anti-apoptotic action of GLP1, we investigated its effect on some of the main regulators of cell apoptosis, including Bcl-2, caspase-3, PARP, and Smac.
Bcl-2 is member of a class of proteins that are mainly localized at the outer mitochondrial membrane and its expression is associated with inhibition of cell apoptosis. Our study demonstrates that in ß-cells transfected with a plasmid encoding for GLP-1 there was a constitutive activation of the expression of Bcl-2. This observation is consistent with studies by Contreras (2001) and Rabinovitch (1999) showing that the overexpression of Bcl-2 confers a long-term protection of islet mass after transplantation (Rabinovitch et al. 1999, Contreras et al. 2001).
All apoptotic pathways, so far identified, converge toward the activation of cytoplasmic cystine proteases named caspases and of the various caspases identified, caspase-3 is one of those that better correlates with cell apoptosis. PARP is involved in the regulation of various important cellular processes such as differentiation, proliferation, and tumor transformation and also in the regulation of the molecular events involved in the recovery of cells from DNA damage (Uchida et al. 2001). In our study, we demonstrated that the exposure of MIN6 cells to immunosuppressive agents was associated with an increased detection of the active form of caspase-3 and an increased production of the p85 subunit of PARP. In cells transfected with GLP-1 the expression of the active form of caspase-3 and of PARPp85 were significantly reduced (Fig. 9).
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Finally, we demonstrated that the capability of GLP-1 transfected MIN6 cells to be resistant to the pro-apoptotic action of immunosuppressive agents was directly mediated by the autocrine production of GLP-1 and it was not due to its effect on the secretion of insulin.
In summary, our results suggest that GLP-1 is a powerful antiapoptotic agent capable of protecting cells from damage induced by immunosuppressive drugs. These observations may have important clinical and therapeutic implications as GLP-1 is being considered as a potential pharmacological agent for the treatment of diabetes.
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Acknowledgements |
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References |
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Received 26 November 2004
Accepted 8 December 2004
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