Anti-inflammatory action of exendin-4 in human islets is enhanced by phosphodiesterase inhibitors: potential therapeutic benefits in diabetic patients
- First Online:
- Cite this article as:
- Pugazhenthi, U., Velmurugan, K., Tran, A. et al. Diabetologia (2010) 53: 2357. doi:10.1007/s00125-010-1849-y
- 654 Downloads
Exendin-4, a glucagon-like peptide-1 (GLP-1) analogue, is reported to have modest anti-inflammatory effects in addition to that of improving beta cell survival. We therefore sought to determine whether exendin-4 decreases expression of the gene encoding chemokine (C-X-C motif) ligand (CXCL)10, which plays a role in initiating insulitis in type 1 diabetes.
The expression of CXCL10 in human islets was determined at the mRNA level by real-time RT-PCR analysis and at the protein level by western blotting. The level of CXCL10 in culture medium was measured by ELISA. Pathway-specific gene expression profiling was carried out to determine the expression of a panel of genes encoding chemokines and cytokines in human islets exposed to cytokines.
IFN-γ induced expression of CXCL10 through activation of signal transducer and activator of transcription-1 (STAT-1). A combination of cytokines (IL-1β, TNF-α and IFN-γ) showed strong synergy in the induction of numerous chemokines and cytokines through nuclear factor kappa B and STAT-1. Exendin-4 suppressed basal expression of several inflammatory mediators. In combination with phosphodiesterase inhibitors, exendin-4 also decreased IFN-γ-induced CXCL10 expression in human islets and in MIN6 cells (a mouse beta cell line), and its secretion into the culture medium. Exendin-4 action was mimicked by forskolin, an activator of adenylyl cyclase, and by dibutyryl cyclic AMP. Protein kinase A was not involved in mediating exendin-4 action on CXCL10. The mechanism of exendin-4’s anti-inflammatory action involved decreases in STAT-1 levels.
These findings suggest that the GLP-1-cyclic AMP pathway decreases islet inflammation in addition to its known effects on beta cell survival.
Cyclic AMP response element binding protein
Chemokine (C-X-C motif) ligand
Chemokine (C-X-C motif) receptor 3
Dibutyryl cyclic AMP
Nuclear factor kappa B
Protein kinase A
Signal transducer and activator of transcription-1
Glucagon-like peptide-1 (GLP-1), an incretin hormone secreted by enteroendocrine L cells of the gut, potentiates glucose-dependent insulin secretion in pancreatic beta cells. GLP-1 activates G-protein coupled receptors, leading to activation of adenylyl cyclase, which results in generation of cyclic AMP. This second messenger plays an important role in mediating the actions of GLP-1, although other signalling pathways are also involved . Several studies have reported the anti-inflammatory action of cyclic AMP . For example, the cytokine-activated janus kinase (JAK)-signal transducer and activator of transcription-1 (STAT-1) signalling pathway is inhibited by cyclic AMP in monocytes, in vascular endothelial cells and in hepatic stellate cells [3–5]. The effects of cyclic AMP are transient because it is degraded by phosphodiesterases. The isozymes of phosphodiesterase play important tissue-specific roles in the regulation of signalling pathways mediated by cyclic AMP. Anti-inflammatory actions of phosphodiesterase inhibitors in NOD mice and in BioBreeding rats have been reported [6, 7].
Chemokines are a family of ∼50 proteins, consisting of 70–130 amino acids that play critical roles in recruitment of leucocytes at the site of inflammation . There are two major families, termed CC and CXC, depending on the arrangement of first two conserved cysteine residues near their N terminus. Chemokine (C-X-C motif) ligand (CXCL)10 is secreted by various cell types, including keratinocytes, intestinal epithelial cells, endothelial cells, monocytes and neutrophils . Diverse biological effects of this highly inducible chemokine have been reported . CXCL10 stimulates T cell adhesion to endothelial cells, suppresses food intake and inhibits angiogenesis . Chemokine (C-X-C motif) receptor 3 (CXCR3), the receptor for CXCL10, is produced in several immune cell types including activated T cells and natural killer cells . CXCL10 plays an important role in trafficking effector T cells to islets during insulitis in type 1 diabetes . In NOD mice, CXCL10 production in islets is increased prior to initiation of insulitis . CXCL10 neutralisation has been shown to suppress the incidence of diabetes in NOD mice . Levels of CXCL10 are elevated in type 1 diabetic patients especially during early and subclinical stages . Islets isolated from type 2 diabetic patients produce high levels of this chemokine . A recent study with pancreatic tissues from four type 1 diabetic patients reported the presence of CXCL10 and infiltration of lymphocytes expressing the corresponding chemokine receptor CXCR3 in islets, regardless of enterovirus infection . These studies suggest that suppression of CXCL10 production in islets could be an important therapeutic approach to reducing insulitis in type 1 diabetes. Recently, in human islets exposed to a combination of proinflammatory cytokines IL-1β, TNF-α and IFN-γ, we observed induction of several chemokines, including CXCL10 . The objective of this study was to determine whether exendin-4-mediated generation of cyclic AMP in human islets leads to suppression of cytokine-induced CXCL10 expression.
Human islets isolated from cadaveric donors were provided by Islet Cell Resource Center Human Islet Basic Science Distribution Program (City of Hope, Duarte, CA, USA). This study was approved by Institutional Ethics Committee. None of the donors had a previous history of diabetes (Electronic supplementary material [ESM] Table 1). Islets with purity of 80% to 95% and viability of 75% to 99% were precultured for 24 h in Miami medium (CMRL 1066 medium [Mediatech, Hendon, VA, USA] supplemented with 0.5% [wt/vol.] human serum albumin and nicotinamide [10 mmol/l]) [18, 19]. Islets were exposed to IL-1β (50 U/ng), IFN-γ (20 U/ng) and TNF-α (100 U/ng) (Roche Applied Science, Indianapolis, IN, USA), and rolipram and cilostamide (Enzo Life Sciences, Plymouth Meeting, PA, USA) or exendin-4 (Sigma Chemical, St Louis, MO, USA). MIN6 cells, a mouse pancreatic beta cell line  obtained from J. Miyazaki (First Department of Surgery, Kyoto University, Japan), were cultured in DMEM containing 5.6 mmol/l glucose, 10% (vol./vol.) FBS, 100 μg/ml streptomycin, 100 U/ml penicillin and 50 μmol/l β-mercaptoethanol at 37°C in a humidified atmosphere with 5% CO2.
RNA isolation and RT-PCR analysis
Total RNA was isolated from treated human islets using an isolation kit (Versagene RNA; Fisher Scientific, Pittsburgh, PA, USA). The mRNA levels of CXCL9, CXCL10 and CXCL11 were measured by real-time quantitative RT-PCR using Taqman probes. The primers and probes (ESM Table 2) were designed by Primer Express (PE ABI, Foster City, CA, USA). Amplicons corresponding to the amplification sequence were synthesised and used as standards in the RT-PCR analysis. mRNA levels of chemokines were expressed in ag/pg of GAPDH.
Western blot analysis
Following treatment, human islets and MIN6 cells were washed with ice-cold PBS and cell lysates were prepared. Western blot analysis was performed by a procedure described previously  using CXCL10 (1:2000; R&D Systems, Minneapolis, MN, USA), phospho STAT-1, STAT-1, phospho cyclic AMP response element binding protein (CREB) or CREB antibodies (1:1000; Cell Signaling, Danvers, MA, USA). After incubation with secondary antibodies conjugated to alkaline phosphatase, signals were developed with CDP-Star reagent (New England Biolabs, Beverly, MA, USA). The membranes were then stripped for 45 min at 55°C in buffer containing 62.5 mmol/l Tris–HCl, pH 6.7, 2% (wt/vol.) SDS and 100 mmol/l β-mercaptoethanol, and reprobed with β-actin antibody (Sigma). The intensity of bands was measured using Fluor-S MultiImager and Quantity One (Bio-Rad, Hercules, CA, USA) and corrected for the levels of β-actin.
ELISA of CXCL10
CXCL10 released into the medium of cultured human islets and MIN6 cells was assayed using kits (DuoSet ELISA Development; R&D Systems) for human or mouse CXCL10 respectively. A 96-well microplate was coated overnight with capture antibody for CXCL10 that was blocked for 1 h at room temperature with 1% (wt/vol.) BSA in PBS. The coated plates were incubated for 2 h at room temperature with samples of medium or CXCL10 standards. After washing, the plates were incubated with biotinylated detection antibody for another 2 h at room temperature. This was followed by incubation with streptavidin conjugated to horseradish peroxidase and colour development with H2O2 and tetramethylbenzidine. The plates were read at 450 nm with wavelength correction at 570 nm. The concentration of CXCL10 was expressed as pg/ml of medium.
Human inflammatory cytokines and receptors PCR array
Functional gene grouping of inflammatory cytokines and receptors array
C5, CCL1–5, CCL7, CCL8, CCL11, CCL13, CCL15-21, CCL23-26, CXCL1-6, CXCL9-14, IL8, IL13
CCR1-9, CX3CR1, IL8RA, XCR1
CD40LG, IFNA2, IL1, IL5, IL8-10, IL13, IL22, IL1A, IL17C, IL1F5-10, LTA, LTB, MIF, SCYE1, SPP1, TNF
IFNA2, IL5RA, IL10RA, IL13RA, IL10RB, IL9, IL13, IL9R
ABCF1, BCL6, C3, C4A, CEBPB, CRP, ICEBERG (also known as CARD18), IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP
ELISA of cyclic AMP
The cyclic AMP levels were determined using a cyclic AMP assay kit (Parameter; R&D Biosystems). In this assay based on competitive binding, cell lysates and cyclic AMP standards are added to a microplate coated with antibody to cyclic AMP. A horseradish peroxidase-labelled cyclic AMP conjugate is also added. Cyclic AMP present in the samples competes with the fixed amount of horseradish peroxidase-labelled cyclic AMP conjugate for sites on the monoclonal antibody. After 2 h incubation, unbound sample and excess conjugate are removed by a wash. A substrate solution is added to the wells to determine bound enzymatic activity. Addition of stop solution halts colour development and absorbance is then measured at 450 nm with correction at 540 nm.
This was performed by one-way ANOVA with Dunnett’s multiple comparison test.
Suppression of IFN-γ-induced CXCR3-binding chemokines by exendin-4 and cyclic AMP in human islets
Decrease of IFN-γ-stimulated production of CXCL10 protein by exendin-4 and cyclic AMP in human islets
Decreased production of STAT-1 by cyclic AMP generation in human islets
Synergistic induction of CXCL10 by cytokines in human islets
Suppression of cytokine-induced CXCL10 expression by cyclic AMP generation in human islets
Suppression of cytokine-induced expression of genes encoding chemokines by inhibitors of NF-kB and STAT-1 pathways, and by cyclic AMP generation in human islets
Suppression of chemokines and cytokines by exendin-4 in human islets
Per cent of control
Per cent of control
Per cent of control
Suppression of cytokine-induced chemokines by cyclic AMP generation and by inhibitors of NF-kB and STAT-1 pathways
Chemokine and cytokine genes
Fold induction over untreated control
Regulation of CXCL10 production by cytokines in MIN6 cells
Suppression of IFN-γ-mediated CXCL10 production by cyclic AMP generation through decrease in the levels of STAT-1 in MIN6 cells
Sustained release of cyclic AMP by exendin-4 in the presence of phosphodiesterase inhibitors in MIN6 cells
Protein kinase A is not involved in cyclic AMP-mediated suppression of CXCL10 levels in MIN6 cells
To delineate the role of PKA-dependent and -independent pathways of cyclic AMP action, we used the PKA inhibitor, H89. Inhibition of PKA decreased cyclic AMP-mediated CREB phosphorylation, as expected (Fig. 8c). However, suppression of IFN-γ-stimulated CXCL10 production by exendin-4 and phosphodiesterase inhibitors was not reversed by H89 (Fig. 8d). This observation suggests that the cyclic AMP-mediated decrease of CXCL10 is unlikely to proceed through PKA. Surprisingly, in human islets, CXCL10 suppression by cyclic AMP was further enhanced by H89 (results not shown), probably via inhibition of a feedback regulation by PKA.
Chemokines play an important role in the initiation of insulitis in type 1 diabetes and in islet graft rejection [12, 13, 23, 24]. In this study, we demonstrate that exendin-4-mediated suppression of CXCL10 expression is enhanced by phosphodiesterase inhibitors in human islets and in a mouse beta cell line. Similar and stronger suppression of CXCL10 was observed in islets treated with: (1) forskolin, which generates cyclic AMP by activation of adenylyl cyclase; and (2) DBC, a non-degradable cyclic AMP analogue. These agents also decreased levels of STAT-1, a transcription factor through which IFN-γ induces CXCL10. PKA does not appear to play a role in mediating the action of cyclic AMP on CXCL10. The anti-inflammatory action of the GLP-1-cyclic AMP pathway was also seen with several other inflammatory mediators generated by human islets. These findings have potential implications for the treatment of diabetes and improvement of islet transplantation outcomes.
IFN-γ was found to be a strong inducer of CXCL10, whereas IL-1β and TNF-α did not increase expression of CXCL10 in human islets (Fig. 4). It is surprising that IL-1β and TNF-α, which are known to induce CXCL10 in other cell types , did not have a significant effect on this chemokine in human islets. However, there was a strong synergy when a combination of these three cytokines was used . The synergy between cytokines could be due to the involvement of multiple transcription factors in the induction of CXCL10. Majumder et al. (1998) have demonstrated the role of STAT-1 and NF-kB in the induction of CXCL10 by IFN-γ and TNF-α . A pathway-specific gene expression profiling analysis revealed induction of a panel of chemokines (CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL20, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL9, CXCL10, CXCL11) and cytokines (IL17C, IL1A, IL1B, IL8 and TNF-α) in human islets following exposure to IL-1β, TNF-α and IFN-γ. The transcription factors NF-kB and STAT-1 seem to be involved in the induction of all these inflammatory mediators except CCL2, CCL7 and IL8. The anti-inflammatory action of exendin-4 and phosphodiesterase inhibitors was seen with CCL2, CCL3, CCL4, CCL7, CCL20, CXCL6, CXCL9, CXCL10, CXCL11, IL1B and IL8. Islet-generated chemokines could play a role in beta cell death in addition to causing insulitis. Impairment of beta cell function in human islets exposed to CXCL10 has been reported .
GLP-1 is known to increase proliferation and neogenesis of beta cells, and to prevent their death by apoptosis . Inhibitors of dipeptidyl peptidase-IV, which is known to increase circulating levels of GLP-1, and degradation-resistant GLP-1 analogues are being used in the treatment of type 2 diabetes to preserve beta cell mass . Anti-inflammatory action of GLP-1 has been suggested by Iwai et al. (2006), who reported the inhibition of lipopolysaccharide-induced IL-1β production in cultured rat astrocytes by GLP-1 through generation of cyclic AMP . Exendin-4 has also been reported to decrease microglia-derived TNF-α and IL-1β in mouse brain . Several in vivo studies have reported the beneficial effects of exendin-4 in NOD mice. For example, chronic administration of exendin-4 to NOD mice leads to modest and significant decrease in insulitis . Continuous infusion of GLP-1 also delayed the onset of diabetes in NOD mice . Other studies have used exendin-4 in combination with anti-inflammatory agents to demonstrate a more significant blocking of insulitis and diabetes onset in NOD mice [31–33]. Furthermore, a recent study has reported that increasing the levels of circulating GLP-1 by inhibiting dipeptidyl peptidase-IV results in prolonged islet graft survival and decreased insulitis in diabetic NOD mice . The mechanism of anti-inflammatory action of exendin-4 could involve downregulation of STAT-1. STAT1 is an autoregulated gene induced by IFN-γ (Fig. 6). Exendin-4 is reported to decrease basal mRNA levels of JAK1 and STAT1 in human islets and in INS-1 cells, a rat insulinoma cell line . We observed here a decrease in IFN-γ-stimulated STAT-1 protein levels pre-incubated with exendin-4 and phosphodiesterase inhibitors (Figs 3c, 7d).
Induction, triggered by viral infection, of CXCL10 expression in islets has been suggested to play a role in initiating insulitis in type 1 diabetes . In a lymphocytic choriomeningitis virus-induced mouse model of autoimmune diabetes, increased expression of CXCR3-binding chemokines was reported as an important early event initiating islet infiltration with immune cells . Cultured human islets have been shown to secrete CXCL10 following enterovirus infection . In a recent report, Tanaka et al. examined sections of pancreas from autopsy material derived from a deceased patient with fulminant type 1 diabetes, an acute subtype of type 1 diabetes identified in the Japanese population, and detected enterovirus capsid protein infiltration of CXCR3-bearing T cells and macrophages . They also observed IFN-γ and CXCL10 in all subtypes of islets. Another recent study has observed the presence of CXCL10 and infiltration of lymphocytes expressing the corresponding chemokine receptor CXCR3 in islets from pancreatic tissues of deceased recent-onset type 1 diabetic patients, regardless of enterovirus infection; this suggests a significant role for this chemokine in inducing insulitis . Viral infection can also cause direct injury to beta cells . Coxsackievirus-induced beta cell death in cultured human islets has been reported . Double-stranded RNA, generated in virus-infected cells, induces the pro-apoptotic gene FAS and synergises with cytokines in inducing apoptosis in rodent beta cells .
The role of inflammatory mediators in type 2 diabetes is also being recognised [42, 43]. The presence of immune cells in islets from human type 2 diabetic patients and from animal models of type 2 diabetes has been reported . Chronic infiltration of diabetic islets with immune cells could play a significant role in beta cell death in type 2 diabetes through release of cytokines and chemokines. Thus our findings of an anti-inflammatory action of exendin-4 have implications for the treatment of both types of diabetes.
Targeting beta cell/islet-generated chemokines could be a potential therapeutic approach along with conventional anti-inflammatory therapy directed against immune cells. Infiltration of islets in type 1 diabetes is preceded by changes in islet milieu . Frigerio et al. have reported that beta cells secrete CXCL10 in response to inflammation . The anti-inflammatory action of the GLP-1-cyclic AMP pathway through decrease of levels of STAT-1 was similar in human islets and MIN6 cells (Figs 3c, 7d). Although exendin-4 did have modest independent anti-inflammatory action in selected experiments (Table 2, Figs 1a, 5b, 7a), addition of phosphodiesterase inhibitors augmented exendin-4 action significantly. Beta cell-specific inhibition of phosphodiesterase could be a potential strategy to sustain the level of cyclic AMP generated by exendin-4. Tissue-specific expression of 11 families of phosphodiesterase isozymes has been reported . Pancreatic beta cells predominantly express phosphodiesterase 3 and phosphodiesterase 4 . Therefore we used cilostamide and rolipram, inhibitors of phosphodiesterase 3 and 4 respectively, to sustain exendin-4-mediated cyclic AMP levels (Fig. 8). The anti-inflammatory action of pentoxifylline, a general phosphodiesterase inhibitor, has also been reported in type 1 diabetic patients  and in animal models of type 1 diabetes [6, 50]. Theophylline, another phosphodiesterase inhibitor, decreases the incidence of diabetes in BioBreeding rats . In the presence of phosphodiesterase inhibitors, exendin-4-mediated phosphorylation of CREB at serine 133, a regulatory step needed for CREB activation, was sustained over time (Fig. 8). Previously, we have reported that CREB is needed for the anti-apoptotic effects of exendin-4 . Thus the present study suggests that a combination of exendin-4 and phosphodiesterase 3 and 4 inhibitors could decrease islet inflammation in addition to improving beta cell mass in patients with diabetes.
This work was carried out with the use of resources and facilities at Denver VA Medical Center. We thank Islet Cell Resource Center-ABCC system and Islet Cell Resource Centers for providing human islets. This work was supported by a grant from American Diabetes Association (1-06-JF-40, to S. Pugazhenthi). We thank M. von Herrath (La Jolla Institute for Allergy and Immunology, San Diego, CA, USA) and R. Mahalingam (University of Colorado Denver, Aurora, CO, USA) for their critical reading of the manuscript.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.