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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.
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Introduction |
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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.
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Materials and Methods |
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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.
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Results |
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). 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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). 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. 2).
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).
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).
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). 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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). 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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). 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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, 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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Discussion |
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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
, 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.
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Footnotes |
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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.
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B. N Friedrichsen, N. Neubauer, Y. C Lee, V. K Gram, N. Blume, J. S Petersen, J. H Nielsen, and A. Moldrup Stimulation of pancreatic {beta}-cell replication by incretins involves transcriptional induction of cyclin D1 via multiple signalling pathways. J. Endocrinol., March 1, 2006; 188(3): 481 - 492. [Abstract] [Full Text] [PDF]
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S. Park, X. Dong, T. L. Fisher, S. Dunn, A. K. Omer, G. Weir, and M. F. White Exendin-4 Uses Irs2 Signaling to Mediate Pancreatic beta Cell Growth and Function J. Biol. Chem., January 13, 2006; 281(2): 1159 - 1168. [Abstract] [Full Text] [PDF]
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 K. Masur, E. C. Tibaduiza, C. Chen, B. Ligon, and M. Beinborn Basal Receptor Activation by Locally Produced Glucagon-Like Peptide-1 Contributes to Maintaining {beta}-Cell Function Mol. Endocrinol., May 1, 2005; 19(5): 1373 - 1382. [Abstract] [Full Text] [PDF]
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E. D'Amico, H. Hui, N. Khoury, U. Di Mario, and R. Perfetti Pancreatic {beta}-cells expressing GLP-1 are resistant to the toxic effects of immunosuppressive drugs J. Mol. Endocrinol., April 1, 2005; 34(2): 377 - 390. [Abstract] [Full Text] [PDF]
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A. K. Bose, M. M. Mocanu, R. D. Carr, C. L. Brand, and D. M. Yellon Glucagon-like Peptide 1 Can Directly Protect the Heart Against Ischemia/Reperfusion Injury Diabetes, January 1, 2005; 54(1): 146 - 151. [Abstract] [Full Text] [PDF]
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P. E. MacDonald, J. W. Joseph, D. Yau, J. Diao, Z. Asghar, F. Dai, G. Y. Oudit, M. M. Patel, P. H. Backx, and M. B. Wheeler Impaired Glucose-Stimulated Insulin Secretion, Enhanced Intraperitoneal Insulin Tolerance, and Increased {beta}-Cell Mass in Mice Lacking the p110{gamma} Isoform of Phosphoinositide 3-Kinase Endocrinology, September 1, 2004; 145(9): 4078 - 4083. [Abstract] [Full Text] [PDF]
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 K. A. Cullen, J. McCool, M. S. Anwer, and C. R. L. Webster Activation of cAMP-guanine exchange factor confers PKA-independent protection from hepatocyte apoptosis Am J Physiol Gastrointest Liver Physiol, August 1, 2004; 287(2): G334 - G343. [Abstract] [Full Text] [PDF]
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 J. F. List and J. F. Habener Glucagon-like peptide 1 agonists and the development and growth of pancreatic {beta}-cells Am J Physiol Endocrinol Metab, June 1, 2004; 286(6): E875 - E881. [Abstract] [Full Text] [PDF]
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P. L. Brubaker and D. J. Drucker Minireview: Glucagon-Like Peptides Regulate Cell Proliferation and Apoptosis in the Pancreas, Gut, and Central Nervous System Endocrinology, June 1, 2004; 145(6): 2653 - 2659. [Abstract] [Full Text] [PDF]
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I. Cozar-Castellano, K. K. Takane, R. Bottino, A.N. Balamurugan, and A. F. Stewart Induction of {beta}-Cell Proliferation and Retinoblastoma Protein Phosphorylation in Rat and Human Islets Using Adenovirus-Mediated Transfer of Cyclin-Dependent Kinase-4 and Cyclin D1 Diabetes, January 1, 2004; 53(1): 149 - 159. [Abstract] [Full Text] [PDF]
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P. E. MacDonald, X. Wang, F. Xia, W. El-kholy, E. D. Targonsky, R. G. Tsushima, and M. B. Wheeler Antagonism of Rat {beta}-Cell Voltage-dependent K+ Currents by Exendin 4 Requires Dual Activation of the cAMP/Protein Kinase A and Phosphatidylinositol 3-Kinase Signaling Pathways J. Biol. Chem., December 26, 2003; 278(52): 52446 - 52453. [Abstract] [Full Text] [PDF]
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D. J. Drucker Glucagon-Like Peptide-1 and the Islet {beta}-Cell: Augmentation of Cell Proliferation and Inhibition of Apoptosis Endocrinology, December 1, 2003; 144(12): 5145 - 5148. [Full Text] [PDF]
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L. Farilla, A. Bulotta, B. Hirshberg, S. Li Calzi, N. Khoury, H. Noushmehr, C. Bertolotto, U. Di Mario, D. M. Harlan, and R. Perfetti Glucagon-Like Peptide 1 Inhibits Cell Apoptosis and Improves Glucose Responsiveness of Freshly Isolated Human Islets Endocrinology, December 1, 2003; 144(12): 5149 - 5158. [Abstract] [Full Text] [PDF]
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 D. J. Drucker Enhancing Incretin Action for the Treatment of Type 2 Diabetes Diabetes Care, October 1, 2003; 26(10): 2929 - 2940. [Abstract] [Full Text] [PDF]
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 U. S. Jhala, G. Canettieri, R. A. Screaton, R. N. Kulkarni, S. Krajewski, J. Reed, J. Walker, X. Lin, M. White, and M. Montminy cAMP promotes pancreatic {beta}-cell survival via CREB-mediated induction of IRS2 Genes & Dev., July 1, 2003; 17(13): 1575 - 1580. [Abstract] [Full Text] [PDF]
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