Glucagon-like Peptide-1Up-regulates Inflammatory Cytokines Expression In Vitro And In Vivo

ObjectiveTo investigate the effect of GLP-1on expression of inflammation cytokines MCP-1and IL-1β in vascular cells, macrophages and pancreatic cells exposed to high glucose in vitro and in diabetic rat aortas in vivo.Methods1. Isolation and culturation of cellsAortic endothelial cells, aortic smooth muscle cells and peritoneal macrophages were all isolated from male Sprague-Dawley rats purchased from animal center, Southern Medical University. Animals were euthanized. For the collection of aortic endothelia cells, the aorta was turned over to expose luminal surface and digested with type I collagenase (2g/L) at37℃for1h. The aorta was cut into pieces and placed luminal side down onto collagen-coated well. For the collection of aortic smooth muscle cells, the aorta was tore off adventitia layer and scrubbed off intima. The media layer was cut into about3x3mm pieces and transferred into6-well plates. Both primary endothelial cells and smooth muscle cells were cultured with DMEM containing20%FBS,100U/ml penicillin and100μg/ml streptomycin. The cells migrated from the aortic rings were grown in containing10%FBS low glucose DMEM for2-3weeks and used for the experiments during passages3-7.For the collection of peritoneal macrophages,10ml serum-free DMEM was injected intraperitoneally and massaged lightly. The peritoneal fluid was withdraw and centrifuged at800r for5min. The pelleted cells were resuspended in DMEM supplemented with10%FBS,100U/ml penicillin and100μg/ml streptomycin, and plated in35-mm dishes. Non-adherent cells were removed after12h by several washes with DMEM, and the macrophages were incubated overnight in the original plating media.Rat INS-1cells (derived from rat endocrine pancreas), human SW1990(derived from human exocrine pancreas) and human PANC-1cells (derived from human pancreatic ducts) were cultured in DMEM supplemented with10%FBS,100U/ml penicillin and100μg/ml streptomycin. All incubations were carried out at37℃in a humidified atmosphere containing5%CO2.2. Type Ⅱ diabetic rat modelMale Sprague-Dawley rats were maintained on a high-fat diet for4weeks. After that, the animals were subjected to an overnight (10h) fast and injected intraperitoneally with STZ. After12h fast, animals showing fasting glucose levels>16.7mmol/l were considered diabetic and used for the study.3. ExperimentsTo measure inflammatory cytokines mRNA treascripts, cells (rat aortic endothelial cells, rat aortic smooth muscle cells, rat peritoneal macrophages, rat INS-1, human SW1990and human PANC-1cells) were grown to75-90%confluence, and followed by starvation in serum-free and glucose-free DMEM medium, then cells were cultured at5.5mM D-glucose (normal glucose, NG) and25mM D-glucose (high glucose, HG) for72h, respectively, in the presence or absence of GLP-1(10nmol/L).The animals were randomly divided into three groups. Group Ⅰ rats were received saline as control. Group Ⅱ rats were given high-fat diet for4weeks and then induced with STZ as diabetes group. Group Ⅲ rats were given high-fat diet for4weeks and treated with GLP-1after2days of being induced with STZ as therapeutic group. Rats were sacrificed after10days and used for subsequent experiments.4. Quantitative RT-PCR analysis of cytokine mRNA expressionThe expression of mRNA for MCP-1and IL-1β mRNA in cultured cells and rat aortas were determined by quantitative RT-PCR. Total RNA was prepared using TRIzol reagent and reverse transcribed using reverse transcription PCR kit. Quantitative PCR was carried out on ABI7500system with SYBR Green. The level of gene expression for each sample was normalized to β-actin mRNA expression using the comparative Ct method.5. Statistical analysisEach experiment was done at least three times and data were expressed as mean±standard deviation (SD). Differences in parameters between multiple groups were evaluated with One-way ANOVA using SPSS13.0software. Differences at P<0.05were considered statistically significant.Results1. In vitro1.1High glucose induced MCP-1and IL-1β expressionMCP-1and IL-1β expression levels in rat peritoneal macrophages were1.5-fold (P=0.014,95%CI-0.260～0.082) and1.3-fold (P=0.033,95%CI-0.234～-0.016) higher, respectively, in high glucose-stimulated than control cells.MCP-1expression levels in rat smooth muscle cells were1.9-fold (P=0.007, 95%CI-0.364～-0.169) higher, in high glucose-stimulated than control cells. The change of MCP-1mRNA expression in rat endothelial cells was not detected.(P=0.083,95%CI-0.001～0.012). IL-1β mRNA expression was not detected under either normal or high glucose condition.MCP-1and IL-1β expression levels in PANC-1cells were2.3-fold (P=0.000,95%CI-0.382～-0.356) and8.2-fold (P=0.000,95%CI-1.057～-0.762) higher, respectively, in high glucose-stimulated than control cells. In rat INS-1and human SW1990cells, neither MCP-1nor IL-1β expression was detected under either normal or high glucose condition.1.2GLP-1up-regulated high glucose-induced MCP-1and IL-1β mRNA expressionTime-course trends of MCP-1were analyzed by RT-PCR and revealed16-fold at4h,12h,24h, and54.8-fold (P=0.000,95%CI1.690-1.786) at48h,69.8-fold (P=0.000,95%CI1.796～1.891) at72h, respectively.In rat peritoneal macrophages, GLP-1induced MCP-1expression levels were34.6-fold (P=0.000,95%CI1.494～1.585) and22.6-fold (P=0.000,95%CI1.308～1.400) higher, respectively, in normal glucose and high glucose-stimulated than control cells. GLP-1induced IL-1β expression levels were94.5-fold (P=0.000,95%CI1.907～2.046) and127.3-fold (P=0.000,95%CI2.038～2.177) higher, respectively, in normal glucose and high glucose-stimulated than control cells.In rat aortic endothelial cells, GLP-1induced MCP-1expression levels were67.3-fold (P=0.000,95%CI1.813～1.842) and65.3-fold (P=0.0000,95%CI1.801～1.830) higher, respectively, in normal glucose and high glucose-stimulated than control cells. IL-1p mRNA was detectable in GLP-1treated cells in either normal or high glucose medium. In rat aortic smooth muscle cells, GLP-1induced MCP-1expression levels were158.2-fold (P=0.000,95%CI1.991～2.404) and114.6-fold (P=0.000,95%CI1.895～2.226) higher, respectively, in normal glucose and high glucose-stimulated than control cells, while IL-1β mRNA was not detectable in GLP-1treated cells in either normal or high glucose medium.In human PANC-1cells, GLP-1induced MCP-1expression levels were1.5-fold (P=0.000,95%CI0.146～0.209) and2.2-fold (P=0.000,95%CI0.300-0.364) higher, respectively, in normal glucose and high glucose-stimulated than control cells. The change of EL-1β mRNA was not detectable in GLP-1treated cells in either normal (P=0.213,95%CI-0.039～0.148) or high glucose (P=0.734,95%CI-0.108～0.079) medium. MCP-1and IL-1β mRNA in rat INS-1and human SW1990cells were not detectable in GLP-1treated cells in either normal or high glucose medium.2. In vivoMCP-1and IL-1β expression levels in diabetic rat aortas were2.2-fold (P=0.000,95%CI0.245～0.421) and5.8-fold (P=0.002,95%CI0.414～1.119) higher, respectively, than control.MCP-1and IL-1β expression levels of rat aortas in therapeutic group were1.6-fold (P=0.001,95%CI0.129-0.306) and2.6-fold (P=0.023,95%CI0.085-0.790) higher, respectively, than diabetes group.Conclusion1. In rat peritoneal macrophages, high glucose induced MCP-1and IL-1β expression, GLP-1up-regulated high glucose induced MCP-1and IL-1β expression.2. In rat aortic endothelial cells, GLP-1induced MCP-1and IL-1β expression levels higher, in normal glucose and high glucose-stimulated than control cells. In rat smooth muscle cells, GLP-1induced MCP-1expression levels higher in normal glucose and high glucose-stimulated than control cells, while IL-1β mRNA was not detectable in GLP-1treated cells in either normal or high glucose medium. 3. In PANC-1cells, MCP-1and IL-1β expression levels were higher in high glucose-stimulated than control cells. In rat INS-1and human SW1990cells, neither MCP-1nor IL-1β expression was detected under either normal or high glucose condition.4. MCP-1and IL-1β expression levels in diabetic rat aortas were higher, respectively, than control. MCP-1and IL-1β expression levels of rat aortas in therapeutic group were higher, respectively, than diabetes group.SignificanceThe present results showed that high glucose stimulated inflammatory factors MCP-1and IL-1β expression in rat peritoneal macrophages, rat smooth muscle cells and pancreatic ductal cells, MCP-1and IL-1β expression levels in diabetic rat aortas were higher, respectively, than control, indicated that inflammation play a certain role in diabetic pathophysiology. GLP-1treatment increased expression of inflammatory factors in these tissues, which suggested that the inflammatory risk should be considered in GLP-1application.