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Endocrinology Vol. 144, No. 4 1444-1455
Copyright © 2003 by The Endocrine Society

ARTICLE

Glucagon-Like Peptide-1 Inhibits Apoptosis of Insulin-Secreting Cells via a Cyclic 5'-Adenosine Monophosphate-Dependent Protein Kinase A- and a Phosphatidylinositol 3-Kinase-Dependent Pathway

Hongxiang Hui, Arash Nourparvar, Xiaoning Zhao and Riccardo Perfetti

Division of Endocrinology and Metabolism (H.H., A.N., R.P.), and Division of Cardiology (X.Z.), Cedars-Sinai Medical Center, Los Angeles, California 90048; and University of California Los Angeles (H.H., R.P.), Los Angeles, California 90024

Address all correspondence and requests for reprints to: Riccardo Perfetti, M.D., Ph.D., Division of Endocrinology, Diabetes, and Metabolism, 8723 Alden Drive, SSB 290, Los Angeles, California 90048. E-mail: perfettir{at}cshs.org.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The activation of the glucagon-like peptide-1 (GLP-1) receptor has been shown to have an important role in the functional activity of islet ß-cells and in the expansion of the islet cell mass. Constant remodeling of islet cell mass is mediated in vivo by proliferative and apoptotic stimuli to ensure a dynamic response to a changing demand for insulin. The present study was undertaken to investigate the biological activity of GLP-1 when cells were challenged by a proapoptotic stimulus. We have shown that activation of the GLP-1 receptor inhibits H2O2-induced apoptosis in a cultured mouse insulinoma cell line, termed MIN6. GLP-1 reduced DNA fragmentation and improved cell survival. This was mediated by an increased expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. GLP-1 also prevented the H2O2-dependent cleavage of poly-(ADP-ribose)-polymerase. Inhibition of the GLP-1-dependent increase of cAMP by Rp-cAMP blocked the antiapoptotic action of GLP-1, as determined by DNA fragmentation and poly-(ADP-ribose)-polymerase assays and by detection of Bcl-2 and Bcl-xL protein levels. Investigation of the role of the protein kinases, PI-3 kinase (PI3K) and MAPK, by use of the inhibitors PD098059 and LY294002 demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H2O2. The present study provides evidence that GLP-1 has an antiapoptotic action mediated by a cAMP- and PI3K-dependent signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
APOPTOSIS, OR PROGRAMMED cell death, is a physiological mode of remodeling tissues during organogenesis and adulthood. The process is characterized by morphological changes, including condensation of the nuclear chromatin, DNA fragmentation, cellular shrinkage, and the formation of apoptotic bodies, which are membrane-bound cellular constituents (1). Apoptotic cell death is an energy-requiring process that involves de novo synthesis of proteins. Various molecules have been demonstrated to regulate, by promoting or inhibiting, the cellular changes that lead to apoptosis. These include the Bcl-2 protein family (2), the caspase family (3), caspase-activated deoxyribonuclease, and inhibitor of caspase-activated deoxyribonuclease (4). Numerous studies have demonstrated that cell apoptosis plays an important role in both the physiological remodeling of the pancreas after birth and pathological pancreatic damage in diabetes (5, 6). Lally et al. (7) and Augstein et al. (8) demonstrated that a great increase of islet cell apoptosis in animal models of type I diabetes correlated with the progression of ß-cell, leading to the onset of hyperglycemia. Recent findings that free fatty acids, glucose, sulfonylurea, and amylin cause ß-cell apoptosis in vitro suggest that apoptosis may also be involved in the pathogenesis of type II diabetes (9, 10). Bonner-Weir and colleagues (11) have shown that, in a Zucker diabetic fatty rat model, the onset of diabetes is caused by an excessive rate of ß-cell death, not by an inefficient replication capacity (11).

In a recent study, we demonstrated that treatment with glucagon-like peptide-1 (GLP-1) drastically reduced the number of apoptotic cells in the pancreas of Zucker diabetic rats. This was indicated by a decrease of terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling-positive-cells, a down-regulation of caspase-3 expression, and an increased expression of Bcl-2 and insulin (12). The present study was undertaken to investigate whether GLP-1 had a direct antiapoptotic effect on insulin-secreting cells, independently from the amelioration of insulin secretion and the acquired glucose control that follow its administration in vivo. We also investigated some of the early events characterizing the signaling pathway that mediates the antiapoptotic action of GLP-1.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mouse insulinoma cell line, MIN6, was a kind gift from Dr. Donald F. Steiner (Howard Hughes Medical Institute, The University of Chicago, IL); Fetal bovine serum (FBS), PBS, penicillin-streptomycin, and cell culture media were obtained from Life Technologies, Inc. (Rockville, MD). The anti-Bcl-xL, anti-Bcl-2, and anti-ß-actin antibody were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Electrochemiluminescence Western blotting detection reagents, Hybond-C nitrocellulose membrane, and cAMP RIA kit were purchased from Amersham Pharmacia Biotech (Denver, CO); X-Omat AR autoradiographic films were from Eastman Kodak Co. (New Haven, CT). H2O2, dimethyl sulfoxide, ethanol, LY294002, PD098059, and Hoechst 33342 were obtained from Sigma (St. Louis, MO); GLP-1, exendin-4, and exendin(9–39)(9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39) were purchased from American Peptide Co. (Sunnyvale, CA); Rp-cAMP was purchased from Calbiochem (La Jolla, CA); Annexin-V-FLUOS staining kit was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Comassie dye assay was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA). The chamber slides for immunostaining were from Nalge Nunc International (Naperville, IL). Fluoromount G was purchased from Electro Microscopy Sciences (Washington, PA).

Cell culture
MIN6 cells were cultured, in 75-ml flasks, in the presence of DMEM with 10% FBS, 100 µg/ml penicillin, 50 µg/ml streptomycin, and 10% fetal calf serum (FCS; Life Technologies, Inc.-BRL) at 37 C under a humidified condition of 95% air-5% CO2. On reaching 80% confluence, the cultures were washed twice with DMEM (without FBS) and kept in serum-free medium for 14 h, before the induction of cell apoptosis. This was obtained by culturing cells in the presence of 50 µM H2O2 for 30 min, in the presence or absence of the indicated peptides or drugs for the specified period of time. After a washout of the cell layer with PBS, adherent cells were scraped off the culture dishes, collected together with detached cells floating in the medium, and spun at 12,000 rpm for 30 sec. Depending on the specific assay for which the cell cultures were prepared, the pellets were either stored at -70 C or used immediately for the experiment.

GLP-1, exendin-4, and exendin(9–39) were diluted in PBS (pH 7.4), whereas dimethyl sulfoxide was used to dissolve Rp-cAMP, LY294002, and PD098059. GLP-1, exendin-4, and exendin(9–39) were solubilized immediately before each individual experiment, whereas other agents were first diluted in stock solutions and stored at -70 C. Control cultures were grown under the same culture conditions as treated cells but in the absence of the drugs. The final concentrations of ethanol and dimethyl sulfoxide were identical, in every culture, irrespective of the particular treatment group.

The concentrations of the following agents were kept constant in all experiments: GLP-1 (10 nM); exendin-4 (10 nM); exendin(9–39) (100 nM); Rp-cAMP (50 µM); LY294002 (50 µM); and PD098059 (50 µM). Each agent was added only once, at the beginning of the individual experiments, with the exception of GLP-1. Fresh aliquots of 10 nM GLP-1 were added every 8 h, to culture medium in all experiments.

SDS-PAGE and Western blot analysis
Cell pellets were lysed at 4 C in buffer containing 60 mM Tris-HCl (pH 6.8), 1% sodium dodecyl sulfate, 10% glycerol, and 0.5% -mercaptoethanol and protease inhibitor mixture (1:100 dilution). Lysates were cleared at 12,000 rpm for 15 min at 4 C and stored at -80 C until needed. Protein concentration was determined using a Comassie dye assay and BSA as a standard. The cell lysates (25–50 µg per sample) were then separated by 8–12% of SDS-PAGE under reducing conditions and electrotransferred onto Hybond-C nitrocellulose membrane 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 working dilutions: Bcl-2 (1:500 dilution); Bcl-xL (1:500 dilution); poly-(ADP-ribose)-polymerase (PARP; 1:500 dilution); and anti ß-actin (1:1000 dilution).

DNA degradation analysis
Floating and adherent cells from each culture condition were combined, centrifuged, pelleted at 400 x g for 5 min, and washed twice with PBS. The pellet was 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 then incubated at 37 C for 2 h. The genomic DNA was extracted by two separations, with phenol/chloroform and then with chloroform only. The DNA pellet was then washed in 70% ethanol and resuspended in 1 mM EDTA, 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.

Hoechst nuclear staining
For morphological studies, the cells were grown in chamber slides and treated with GLP-1 and H2O2 as described above. They were then washed in PBS (pH 7.4) and fixed for 20 min in 2% paraformaldehyde in PBS (pH 7.4) at room temperature. After a wash in PBS, the cells were permeabilized in 0.1% Triton X-100 in 0.1% sodium citrate, rinsed twice in PBS, and stained with the karyophilic dye Hoechst 33342 (10 µg/ml) for 5 min at room temperature. After a final wash in PBS, the cells were mounted in Fluoromount G, and visualized under UV light with an Axiophoto microscope (Carl Ziess, New York, NY).

FACS analysis for Annexin-V
Apoptotic cells were analyzed by flow cytometer (FACS; Becton Dickinson and Co., San Jose, CA); using the Annexin-V-Flous Staining kit. Annexin-V is a Ca2+-dependent phospholipid-binding protein with high affinity for phosphotidylserine (PS); hence, this protein can be used as a sensitive probe for PS exposure on the outer leaflet of the cell membrane and be used for the detection of apoptotic cells. Apoptotic cells have then to be differentiated from necrotic cells, because these cells also expose PS because of the loss of membrane integrity. The simultaneous application of propidium iodide as a DNA stain, used for dye exclusion tests, allows Annexin-V positively stained cell cluster to be distinguished from necrotic cells.

For this study, the dose of H2O2 was reduced to 20 µM. This change in the experimental protocol was introduced to compensate for the much greater sensitivity of assays detecting for Annexin-V (an early marker of cell apoptosis), when compared with the other techniques to evaluate cell apoptosis that are employed in this study. Cells were collected from the culture flasks and washed twice with PBS by centrifugation at 200 x g for 5 min. The cell pellet was then suspended in 100 µl 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), using the LYSIS II analyzer program.

cAMP assay
Cells were washed twice with cold PBS, and cellular cAMP was then extracted by a liquid phase extraction method. Briefly, cell pellets were suspended and washed in 65% ethanol (kept at -20 C before use). After the cells were lysed, the supernatant was collected in a new tube, then evaporated under a vacuum oven. The dried extracts were dissolved in 100 µl assay buffer before analysis. The RIA for cAMP was performed after an overnight protocol, as described by the manufacturer. Total protein concentration for each individual sample was used to normalize the concentration of cellular cAMP.

Statistical analysis
The data were expressed as mean ± SE. Comparison of individual treatments was conducted using Student’s t test. Data analysis showing statistical significance was further evaluated by Dunnett’s post hoc test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
GLP-1 protects insulin-secreting cells (MIN6) from H2O2-induced apoptosis
We first examined the morphological changes of MIN6 cells when challenged with the proapoptotic agents H2O2. Nuclear staining with the dye Hoechst 33342 demonstrated that, whereas the exposure of cells in culture exposed to H2O2 promoted changes in the nuclear morphology characteristic of apoptotic cells, the treatment with GLP-1 before the exposure to H2O2 was capable of preventing the nuclear fragmentation and inhibiting cell apoptosis (Fig. 1Go). Interestingly, GLP-1 had an antiapoptotic effect only when cells were pretreated with this agent. Exposure to GLP-1 immediately (1–2 min) after cells were treated with H2O2 had no effect on nuclear changes leading to apoptosis and ultimately on cell survival (data not shown).



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  		Figure 1. GLP-1 protects insulin-secreting cells (MIN6) from H2O2-induced apoptosis. Cells were exposed to H2O2, in the presence or absence of GLP-1. After fixing the cells, they were stained for the nuclear marker Hoechst 33342. Apoptotic cells were identified by their typical nuclear appearance. A, Control MIN6 cells cultured in regular medium; B, MIN6 cells cultured in medium containing GLP-1 (10 nM for 16 h); C, MIN6 cells exposed to H2O2 (50 µM for 30 min); D, MIN6 cells pretreated with GLP-1 (10 nM for 16 h), then exposed to H2O2 (50 µM for 30 min). The graph on the lower left quadrant shows the percentage of apoptotic cells in each culture condition. At least 10 fields per dish and 4 independent cultures were examined. Arrows in C and D indicate examples of apoptotic cells. The inset in C shows a detail of the morphological changes of the nuclei of two apoptotic cells.

 
GLP-receptor agonists protect cells from H2O2-dependent DNA degradation
Activation of a calcium-dependent endonuclease is one of the earliest detectable events denoting an irreversible commitment to cell death. The endonucleases attack the DNA structure producing the formation of DNA fragments of 180–200 bp. Electrophoresis of genomic DNA from MIN6 cells that were exposed to H2O2 showed the characteristic laddering pattern that leads to cell death (Fig. 2Go). DNA fragmentation was inhibited by treatment with GLP-1 before exposure to H2O2. Specificity of the antiapoptotic action of GLP-1 was demonstrated by the lack of antiapoptotic activity of the GLP-1-receptor antagonist exendin(9–39), and by the inhibition of DNA fragmentation observed with the receptor agonist exendin-4 (Fig. 2Go).



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  		Figure 2. GLP-1 inhibits DNA fragmentation resulting from H2O2- induced apoptosis. MIN6 cells were exposed to H2O2 (50 µM for 30 min) in the presence of the following GLP-1 receptor ligands: GLP-1 (10 nM); exendin (Ex)-4 (10 nM); Ex-9 (100 nM); and a combination of both GLP-1 and Ex-9 (10 nM and 100 nM, respectively). GLP-1 receptor ligands were added to the culture medium 16 h before adding H2O2. The blot presented is representative of three independent experiments.

 
GLP-1 inhibits PS translocation leading to cell apoptosis
A characteristic biochemical change that is associated with cell apoptosis, such as the translocation of PS from the inner leaflet to the outer leaflet of the plasma membrane, was also investigated. We stained MIN6 cells with Annexin-V-fluoroscein isothiocyanate, and then subjected the cells to FACScan analysis. The intensity of fluorescence of Annexin-V-fluoroscein isothiocyanate-stained cells is directly proportional to the amount of PS on the outer leaflet of the plasma membrane. The results indicate that 8 ± 5%; 6 ± 2%; 80 ± 4% and 15 ± 5% of the MIN6 cells were Annexin-V-positive when they were grown in regular medium (for 16 h), cultured in the presence of GLP-1 (10 nM for 16 h) treated with H2O2 (20 µM for 30 min), or pretreated with GLP-1 (10 nM for 16 h) then exposed to H2O2 (20 µM for 30 min), respectively (Fig. 3Go).



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  		Figure 3. GLP-1 prevents the early events associated with cell apoptosis. FACS analysis for Annexin-V. A, Control MIN6 cells were cultured in regular medium; B, cells cultured in medium containing GLP-1 (10 nM for 16 h); C, cells exposed to H2O2 (20 µM for 30 min); D, MIN6 cells pretreated with GLP-1 (10 nM for 16 h), then exposed to H2O2 (20 µM for 30 min). The top part of the figure represents the average of three independent experiments for culture condition, whereas the bottom part shows a representative FACS analysis for the four different culture conditions studied. For each FACS report, the lower left quadrant is where living cells were detected; in the upper right quadrant, necrotic cells; and the lower right quadrant, apoptotic cells. NS, Not significant.

 
GLP-1 increases the expression of cellular antiapoptotic proteins
The observation that GLP-1 was capable of protecting cells from H2O2-induced apoptosis only when it was administered before H2O2, but not capable of rescuing cells after H2O2 has initiated the cascade of events leading to cell death, suggests that GLP-1 may act by enhancing the expression and/or activity of endogenous antiapoptotic factors. Western blot analysis for the antiapoptotic proteins Bcl-2 and Bcl-xL showed that their abundance was increased when cells were preexposed to GLP-1 (Fig. 4Go).



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  		Figure 4. GLP-1 increases the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. A, Western blot analysis for Bcl-2, Bcl-xL, and ß-actin of MIN6 cells exposed to H2O2, in the presence or absence of GLP-1. Lane 1, Control MIN6 cells cultured in regular medium; lane 2, MIN6 cells exposed to H2O2 (50 µM for 30 min); lane 3, MIN6 cells pretreated with GLP-1 (10 nM for 16 h) and then exposed to H2O2 (50 µM for 30 min). Lanes 4 and 5 show a representative Western blot analysis of cells nonexposed to H2O2 and analyzed for Bcl-2, Bcl-xL, and ß-actin levels. Lane 4, Control MIN6 cells cultured in regular medium; lane 5, MIN6 cells treated with GLP-1 (10 nM for 16 h). The blot presented is representative of three independent experiments. The graphs on the bottom represent the average of four independent Western blotting experiments for Bcl-2 (B) and Bcl-xL (C). Bcl-2 and Bcl-xL protein levels were normalized for ß-actin level and are shown in the graphs as fold difference, compared with control (=1).

 
Measurement of p85 PARP
PARP is a DNA repair enzyme whose proenzyme form is cleaved by caspase 3 in certain cells that are undergoing apoptosis. Western blot analysis revealed minimal cleavage of the proenzyme form of PARP in cells that were pretreated with GLP-1 before exposure to H2O2 (Fig. 5Go). In comparison, cells that were cultured in the presence of the GLP-1 receptor antagonist exendin(9–39) and then exposed to H2O2 showed a marked expression of the p85 cleaved fragment, whereas cells cultured with the agonist exendin-4 were resistant to the proapoptotic action of H2O2 (data not shown).



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  		Figure 5. GLP-1 inhibits the cleavage of PARP in response to H2O2. MIN6 cells exposed to H2O2, in the presence or absence of GLP-1, analyzed by immunoblot with antibody to PARP. Lane A, Control MIN6 cells cultured in regular medium; lane B, MIN6 cells exposed to H2O2 (50 µM for 30 min); lane C, MIN6 cells pretreated with GLP-1 (10 nM for 16 h) and then exposed to H2O2 (50 µM for 30 min). Lane D shows a protein extract obtained from MIN6 cells treated with GLP-1 (10 nM for 16 h) and not exposed to H2O2. The blot presented is representative of three independent experiments.

 
GLP-1 antiapoptotic action is mediated by the activation of a cAMP-dependent and a PI-3 kinase (PI3K)-dependent signaling pathway
Most, although not all, of the activities initiated by binding of GLP-1 to its receptor on the cell surface of insulin-secreting cells are mediated by an increase in intracellular cAMP level. We therefore investigated whether cAMP was also involved in the regulation of the antiapoptotic action of GLP-1. We observed that the antiapoptotic action of GLP-1 was significantly reduced by blocking cAMP production with Rp-cAMP, as determined by DNA fragmentation and PARP assays (Fig. 6Go). Western blot analysis for the antiapoptotic proteins Bcl-2 and Bax-xL demonstrated that Rp-cAMP greatly reduced the GLP-1-dependent regulation of their cytoplasmic levels.



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  		Figure 6. Effect of cAMP inhibition on the antiapoptotic action of GLP-1. Cells were exposed to H2O2 (50 µM for 30 min), in the presence or absence of GLP-1 (10 nM; added 16 h before H2O2). GLP-1 antiapoptotic activity was tested in the presence of the cAMP-dependent protein kinase (PKA) inhibitor Rp-cAMP. A, DNA fragmentation assay; B, PARP cleavage assay; C, Western blot for Bcl-2, Bcl-xL, and ß-actin. Graphs in D and E represent the Bcl-2 and Bcl-xL levels derived from three independent experiments. Bcl-2 and Bcl-xL protein levels were normalized for ß-actin level and are shown in the graphs as fold difference, compared with control (=1).

 
GLP-1 has also been demonstrated to operate via a kinase-dependent signaling pathway in the regulation of the gene transcription for insulin and other ß-cell-specific genes. Considering this evidence, we investigated the role of the protein kinases PI3K and MAPK in the prevention of apoptosis and in the regulation of antiapoptotic proteins. The use of the inhibitors PD098059 and LY294002 (which are capable of selectively blocking MAPK and PI3K, respectively) demonstrated that the activation of PI3K, but not MAPK, was required to prevent proapoptotic events in cells exposed to H2O2 (Fig. 7Go). LY294002, but not PD098059, prevented the capability of GLP-1 to block the DNA fragmentation induced by H2O2. Cleavage of PARP by caspase-3, characteristic of cells undergoing apoptosis, was also observed in cells where LY294002 was added to GLP-1. Finally, LY294002 blocked the antiapoptotic increase in Bcl-2 and Bcl-xL that was induced by GLP-1.



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  		Figure 7. The antiapoptotic action of GLP-1 requires the activation of a PI3K-dependent signaling pathway. Cells were exposed to H2O2 (50 µM for 30 min), in the presence or absence of GLP-1 (10 nM; added 16 h before H2O2). GLP-1 antiapoptotic activity was tested in the presence of the PI3K inhibitor LY294002 and the MAPK inhibitor PD098059. A, DNA fragmentation assay; B, PARP cleavage assay; C, Western blot for Bcl-2, Bcl-xL, and ß-actin. Graphs in D and E represent the Bcl-2 and Bcl-xL levels derived from three independent experiments. Bcl-2 and Bcl-xL protein levels were normalized for ß-actin level and are shown in the graphs as fold difference, compared with control (=1).

 
To study whether the activation of cAMP and PI3K resulted from the activation of a single GLP-1-dependent antiapoptotic pathway, we investigated whether, by blocking either one of them, we were able to abolish the overall effect of GLP-1 on cell survival. For this study, cells were cultured in the presence of the cAMP inhibitor Rp-cAMP or LY294002, or finally the two agents together, and analyzed by FACS analysis for Annexin-V. As shown in Fig. 8Go, the inhibition of either cAMP or PI3K did not entirely abolish the effect of GLP-1, suggesting that the two signaling pathways had a cooperative, but largely independent, mechanism of activation. Interestingly, GLP-1 had an antiapoptotic effect that was greater than the one derived from the sum of the PI3K-dependent and cAMP-dependent effect, suggesting either a cross-talk between the two, with a final additive effect, or the potential existence of addition signaling pathways involved in this biological action.



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  		Figure 8. The antiapoptotic action of GLP-1 is mediated by the independent activation of a PI3K-dependent and a cAMP-dependent pathway. Cells were exposed to H2O2 (50 µM for 30 min) and cultured in the presence of GLP-1 (10 nM; added 16 h before H2O2), with or without the cAMP-dependent protein kinase (PKA) inhibitor or PI3K inhibitor LY294002. They were then analyzed by FACS analysis for Annexin-V. In each panel, the lower left quadrant indicates the living cells, whereas the lower right shows the apoptotic cells. A, Control MIN6 cells cultured in regular medium; B, cells exposed to H2O2 alone; C, cells exposed to GLP-1 and H2O2; D, cells exposed to GLP-1, Rp-cAMP, and H2O2; E, cells exposed to GLP-1, LY294002, and H2O2; F, cells exposed to GLP-1, LY294002, and Rp-cAMP. A–F represent individual experiments, and Table 1Go represents the average of five independent experiments. FL, Fluorescence.

 
The use of protein kinase A (PKA) and PI3K inhibitors provided also some preliminary data indicating that the signaling pathway(s) mediating the antinecrotic activity of GLP-1 differs from those used for its antiapoptotic action. Indeed, the single blockade of PKA or PI3K pathway did not interfere with the protective action of GLP-1 against H2O2-induced cell necrosis. On the other hand, the antinecrotic action of GLP-1 was reversed by the simultaneous exposure of cells to both PKA and PI3K signaling pathway inhibitors (Table 1Go).


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  		Table 1. Necrotic, living and apoptotic cells under different treatment condition

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The regulation of islet cell mass involves the integrated functions of ß-cell proliferation and neogenesis, on one hand, and of ß-cell destruction or apoptosis, on the other hand. Factors that can augment ß-cell mass are of particular interest in the field of islet cell transplantation, because there are insufficient quantities of islets available for the number of recipient candidates (13, 14). A proproliferative action of GLP-1 on insulin-secreting cells has been shown in both in vitro and in vivo models (15). In the present study, we show, for the first time, that GLP-1 has antiapoptotic properties in addition to its effect on the expansion of islet cell mass.

Prior observations from our laboratory have shown that GLP-1 reduced the number of apoptotic cells in the pancreas of Zucker diabetic rats (12). To investigate whether this observed decrease in apoptosis is independent of the effects of GLP-1 on glucose control and insulin secretion, we used a MIN6 insulinoma cell line in vitro. We were able to demonstrate that GLP-1 prevents apoptosis in MIN6 cells exposed to oxidative damage via H2O2 but is unable to rescue cells from apoptosis once such damage has proceeded. This observation led to the hypothesis that GLP-1 acts by induction of cellular antiapoptotic proteins or by enhancing their expression or activity. We then opted to elucidate the molecular mechanism/signaling pathway through which GLP-1 exerts its antiapoptotic effect.

It has been confirmed that both direct cytotoxic T-cell- and indirect cytokine nitric oxide- or free radical mechanisms, as well as viruses, can induce ß-cell apoptosis in vitro (16, 17). Reactive oxygen species (ROS) are presumed to be one of the important regulators of apoptosis and has been observed to be in most diabetes cases. Production of ROS is found to be stimulated by TNF-, lipopolysaccharide-, ceramide-, growth factor withdrawal-, human immunodeficiency virus infection-, or p53-induced apoptosis (18, 19). In contrast, overexpression of thioredoxin, manganese superoxide dismutase, or Bcl-2 can delay apoptosis (20, 21). H2O2 is one of the most powerful oxidizers; and, through catalysis, it can be converted into hydroxyl radicals, one of the main forms of ROS (22). It has been shown that high doses of H2O2 can cause cell necrosis; whereas in low doses, it induces apoptosis of many cultured cell lines. The latter occurs via an increased level of intracellular Ca2+, a down-regulation of GSH (reduced glutathione) levels, and an increased lipid peroxidation. These events lead to a change in the ratio of reduced ion components to oxidized cellular ions (23). In the present study, we showed that MIN6 cells cultured in the presence of 20–50 µmol H2O2 were capable of inducing cell apoptosis, as demonstrated by FACS analysis for Annexin-V, internucleosomal DNA fragmentation, DNA-laddering, and activation of PARP. We also observed that H2O2 decreased the expression of the antiapoptotic proteins Bcl-2 and Bcl-xL. Based on these data, we used H2O2 as a model to study ß-cell apoptosis and to study the putative antiapoptotic action of GLP-1.

Many growth factors, characteristically known for their proproliferative properties and/or their action on cell differentiation, have also been shown to be capable of interfering with the sequence of events leading to cell apoptosis. The following growth factors have been investigated on their ability of inducing, delaying, or preventing programmed cell death in vitro: TGF{alpha}, TGFß1, brain-derived neurotrophic factor, acidic fibroblast growth factor, basic fibroblast growth factor, IGF-1, platelet-derived growth factor, and hepatocyte growth factor (24, 25, 26). In the present study, we demonstrated that, whereas the addition of GLP-1 to cells exposed to H2O2 was not capable of rescuing them from apoptosis, the administration of GLP-1 before the exposure to H2O2 had a significant protective effect against cell apoptosis. This finding was supported by FACS analysis for Annexin-V, morphological changes of the nuclear appearance, and DNA laddering. To investigate whether the antiapoptotic action of GLP-1 was both specific and a GLP-1 receptor-mediated event, cells were also cultured in the presence of the GLP-1 receptor agonist exendin-4, as well as the antagonist exendin(9–39). Whereas exendin-4 had an antiapoptotic action, exendin(9–39) inhibited the GLP-1-dependnet protection against the H2O2- induced apoptosis of MIN6 cells.

Interestingly, a similar antiapoptotic effect of GLP-1 has been recently proposed for cultured hippocampal neuronal cells that were exposed to the proapoptotic action of glutamate (27).

In studying factors that could have led to the prosurvival action of GLP-1, we investigated its effect on some of the main regulators of cell apoptosis, the Bcl-2 family proteins (28). This class of proteins is represented by molecules that are mainly localized at the outer mitochondrial membrane and can be both prosurvival and proapoptotic modulators. The prosurvival members are represented by Bcl-2 and Bcl-xL (29, 30), whereas the proapoptotic proteins include Bax, Bad, and Bid (31). Our experiments showed that GLP-1 increased the expression of Bcl-2 and Bcl-xL in MIN6 cells that were treated with GLP-1 before being challenged with H2O2.

PARP modifies various nuclear proteins by poly-(ADP-ribosyl)ation; its modification 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 (32). Furthermore, PARP inhibits Ca 2+/Mg2+-dependent endonucleases that cleave DNA during apoptosis. In apoptosis cascade, caspases 3, 6, 7, 8, and 9 can inactivate PARP by cleaving the molecule into two pieces, as shown in our H2O2-induced apoptotic MIN6 and other apoptotic models (33). In our experiment, after MIN6 cells were pretreated with GLP-1, the cleavage of PARP was inhibited. This further demonstrated that GLP-1 greatly prevents the apoptosis induced by H2O2 in MIN6 cells. All these observations lead to the conclusion that GLP-1 acts by induction of cellular antiapoptotic proteins or by enhancing their expression or activity.

GLP-1 regulates insulin secretion via a cAMP- and Ca2+-dependent signaling pathway (34). Several studies aimed at inhibiting GLP-1-dependent regulation of cAMP have been performed to investigate whether the same signaling pathway was also involved in its antiapoptotic action. Indeed, an increase in cellular cAMP level has been shown to modulate apoptosis in various cell types. This includes the regulation of apoptosis in granulosa cells of rat and human ovary (35), as well as in development regulation in thymocytes and mature B cells (36). On the other hand, there are experimental data showing that agents capable of increasing intracellular cAMP protect against apoptosis induced by hydrophobic bile acids (37). In the present study, the apoptotic protection exerted by GLP-1 in MIN6 cells challenged with H2O2 was abolished by Rp-cAMP [a cAMP-dependent protein kinase (PKA) inhibitor], suggesting that cAMP is a positive mediator in the prevention of apoptosis of insulin-secreting cell lines.

PI3K and MAPK are two important signaling molecules mediating cell proliferation, differentiation, and apoptosis (38). Several studies have shown that activation of PI3K is required for the antiapoptotic effect of NGF, GLP-2, 8-(4-chlorothiophenyl) cAMP, and hepatocyte growth factor in some models of apoptosis in vitro (39, 40). Our study, using MIN6 cells, indicated that LY294002 inhibited the antiapoptotic activity of GLP-1, suggesting that this was partially regulated via a PI3K- dependent signaling mechanism. This is consistent with the observations that expression of a constitutively active PI3K prevents the activation of caspase 3 and apoptosis of cardiac muscle cells, and that suppression of transforming growth factor-ß induced apoptosis through a phosphatidylinositol 3-kinase/Akt-dependent pathway (41). In conjunction, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis.

The MAPK pathways transduce a variety of external signals, leading to a wide range of cellular responses, including growth, differentiation, inflammation, and apoptosis (42). In mammals, three major MAPK pathways have been identified: p44/42 MAPK/ERK (extracellular signal-regulated protein kinase), SAPK/JNK (c-Jun N terminal kinase/stress-activated protein kinase), and p38 MAPK. The MAPK/ERK signaling cascade is activated by a wide variety of receptors involved in growth and differentiation, including receptor tyrosine kinases, integrins, and ion channels (42). In addition, GLP-1, gastric inhibitory peptide, secretin, pituitary adenylyl-cyclase-activating protein, and vasoactive intestinal polypeptide (all peptide hormones that activate receptors coupled to the production of cAMP) have been shown to activate MAPK/ERK (43, 44, 45, 46, 47, 48). An activated ERK dimer can regulate targets in the cytosol and also translocate to the nucleus, where it phosphorylates a variety of transcription factors regulating gene expression. In our study, the use of the p44/42 MAPK inhibitor PD098059, together with GLP-1, had no significant effect on H2O2- induced apoptosis, indicating that MAP/ERK signaling was not involved in the protection of cells from apoptosis in the in vitro system that we used. This observation shares some similarity with studies conducted to characterize the antiapoptotic action of GLP-2, a peptide hormone with a significant similarity to GLP-1, which has also been shown to protect cells from apoptosis via a MAPK-independent pathway (49). Our study does not rule out that other MAPK pathways may be involved in the antiapoptotic action of GLP-1, and studies directed at addressing this question more directly may need to be carried out to further elucidate the signaling molecules mediating the GLP-1-dependent protection from cell death.

It has been reported that cAMP-mediated cytoprotection against bile acid-induced apoptosis involves PKA, MAPK, and PI3K (37). It has also been shown that signaling through PI3-K and a Ser/Thr kinase, Akt/PKB may protect against apoptosis independently from cAMP (50). To study the relationship between cAMP-dependent and PI3K-dependent pathways for GLP-1 antiapoptotic activity in insulin-secreting cells, we compared the inhibition efficiency of Rp-cAMP and LY 294002, as well as the putative cooperative effect of the two agents together in the inhibition of GLP-1 action. We demonstrated that cAMP and PI3K independently mediated the action of GLP-1. In addition, culturing cells with the two agents together produced a proapoptotic effect greater than the sum of the two agents individually.

In addition to the effect of GLP-1 on apoptosis, studies of H2O2-mediated cell death showed that, in addition to preventing cell apoptosis, GLP-1 had a protective effect against cell necrosis. Interestingly, in studying the contribution of PKA and PI3K signaling pathways to the antinecrotic effect of GLP-1, we found that there were some differences, when compared with its antiapoptotic effect. Indeed, when PKA or PI3K inhibitors were used individually, they seemed to be unable to prevent the positive effect of GLP-1 on cell necrosis. This differed from the result observed when both PKA and PI3K signaling pathways were blocked simultaneously. The latter led to the necrotic death of approximately one third of the cells in culture, and this was similar to the percentage of necrotic cells detected in our positive control culture treated with H2O2 alone. This data seems to indicate that a converging mechanism requiring both PKA and PI3K signaling needs to be activated to avoid cell necrosis, as induced in vitro, by H2O2.

In summary, our results suggest that GLP-1 is not only a growth factor for ß-cells but also a powerful antiapoptotic agent contributing to the observed increase in islet cell mass in prior in vivo models. These observations may have important clinical and therapeutic implications as GLP-1 is being considered both for the treatment of type-2 diabetes mellitus as well as for pancreatic islet transplantation.


   Footnotes
 
This work was supported by the Foundation for Diabetes Research.

Abbreviations: FBS, Fetal bovine serum; GLP-1, glucagon-like peptide-1; PARP, poly-(ADP-ribose)-polymerase; PI3K, PI-3 kinase; PKA, protein kinase A; PS, phosphotidylserine; ROS, reactive oxygen species; Rp, R-isomer of the phosphorus chiral center.

Received August 30, 2002.

Accepted for publication December 3, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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