The Effect Of GLP-1on Islet Microvascular Endothelial Cells And Islet Function Injuried By Lipotoxcity

Backgrounds and aims:In2009, the incidence of diabetes in China's urban population reached9.7%, which indicated that the number of Chinese patients with diabetes has reached92.4million and the prevention work is arduous. More than90%of the patients suffered to type2diabetes. The main mechanism of type2diabetes is insulin resistance and the decline of pancreatic β cell function, while the latter is the central link of type2diabetes. The main pathological mechanisms that cause pancreatic β cell dysfunction is lipotoxicity and glucose toxicity. With the great changes in the structure of the daily diet of the Chinese population, a lot of fatty foods intake, lipid metabolism caused by excess fat toxic injury played a decisive role in the incidence of type2diabetes in progress.Oxidized low-density lipoprotein (Ox-LDL) is one of the most important risk factors of metabolic disease, particularly diabetes. Several studies demonstrated that Ox-LDL induces early endothelial dysfunction in cultured micro-and macrovascular endothelium, characterized by a series of changes in proliferation, barrier function, sensitivity to apoptosis and adhesion, and angiogenic as well as synthetic properties of endothelial cells (ECs). However, studies of islet-derived ECs are lacking. In light of the endothelial-endocrine axis within adult pancreatic islets, Ox-LDL may induce alterations in islet endothelium, potentially contributing to the progressive reduction of β-cell mass and function that characterizes the natural history of type2diabetes. Recent studies indicate that the islet vasculature likely plays a role in the physiology and pathophysiology of the pancreatic islets. Besides providing oxygen, nutrients, and secretory signals from other cells to endocrine cells and producing a number of vasoactive, angiogenic substances, cytokines, and growth factors, islet endothelium induces insulin gene expression during endocrine tissue development to promote β-cell proliferation and affect adult β-cell function.Increasing evidence shows that apoptosis of islet microvascular endothelial cells (IMECs) participates in the pathogenesis of diabetes. The pancreatic endocrine vasculature has distinctive functional and structural features, which renders it highly adapted to communicate with the underlying endocrine tissue in a cross-talk relationship. However, the microvasculature has a key role in the interface between the vascular space and organ parenchymas and participates in numerous pathophysiological processes. Pancreatic islets are one of the most vascularized organs,. The islets of Langerhans consist of endocrine cells embedded in a network of specialized capillaries that regulate islet blood flow. Pancreatic islet microcirculation has distinctive high permeability, with an islet capillary network showing5times higher density than the capillary network of the exocrine counterpart.The incretin hormone glucagon-like peptide-1(GLP-1) is a30-amino acid peptide that is secreted from the intestinal L cells in response to nutrient ingestion,. It stimulates glucose-dependent insulin production and secretion from(3cells and inhibits glucagon secretion. This peptide possesses various actions that are protective against diabetes and are mediated at the level of the (3cell, as well as in peripheral tissues.Although,conventional C57BL/6mice can make insulin resistance (IR) after long-term high fat diet, but it can not be induced into significant hyperglycemia, hypercholesterolemia and atherosclerosis, which is still different with the clinical features of human T2DM. ApoE-/-C57BL/6mouse characterized by the genetic background of dyslipidemia and atherosclerosis.Under long-term high-fat diet,ApoE-/-mice fasting plasma glucose and plasma insulin levels increased. In this study, we used ApoE-/-mice as a basic animal models, given the high-fat diet to establish the model of the lipotoxicity islet dysfunction.Therefore, the aims of this study were as follows:1. Set up Lipotoxicity-islet microvascular endothelial cells injury model to explore the impact of GLP-1on islet microvascular endothelial cells through PARP-1/iNOS/NO pathway.2. Construction of PLVX-AcGFP-N1-PARP-1lentiviral vector and its expression in MS-1cells.3. Set up ApoE-/-mouse islet injury model under high fat diet, further validation the influence of GLP-1to pathway islet function by PARP-1/iNOS pathwayMethodsPart I:Glucagon-like peptide1protects microvascular endothelial cells by inactivating the PARP-1/iNOS/NO pathwayFor dose-dependent effects, MS-1cells were treated with0,10,30,50,100and200μg/mL Ox-LDL48h before cells were harvested for measurements of target gene mRNA and protein levels.To evaluate the apoptosis situation of MS-1by MTT reduction cytotoxicity assay. Obaerve the mRNA and protein expression of PARP-1and iNOS by Quantitative real-time PCR and Western blot. Nitrite concentrations were then determined from a calibration curve of standard NaNO2concentrations against absorbance. MS-1cells were fixed with4%paraform/PBS, then underwent TUNEL assay with the one-step TUNEL apoptosis assay kit.Part II:Construction of Lentiviral vectors PLVX-AcGFP-N1-PARP-1and Its Expression in MS-1CellsUsing Gene coding method, a cassette consisting of human PAPR-1DNA was fused by a linker coding for sequesnes rich in glycine by PCRtechnique and cloned into PLVX-AcGFP-N1. The plasmid PLVX-AcGFP-N1-PARP-1and the other two packaging plasmid were co-transfected into293FT cells. The viral supernatant was collected. The viral titer was detected and the expression of usion gene encoding PARP-1was detected in the MS-1cell lines treated with PLVX-AcGFP-N1-PARP-1.Part III:Lipotoxity injury islet function in ApoE-/-mice and GLP-1interventionHealthy male ApoE-/-mice, eight-week-old, were randomly divided into normal-chow diet fed group (NF group, n=30) and high-fat diet fed group (HF group, n=40) and fed with corresponding food for0,4,8,16,32weeks. In addition, Normal food mice were set as control group(NF group, n=10). The Blood glucose and lipid metabolism in awake mice were evaluated by tail blood. Then male ApoE-/-mice were randomly divided into6groups.The mice were given an intraperitoneal injection of DPQ(PARP-1inhibitor group,PARP-1lentivirus (PARP-1overexpress group, GLP-1group,80μg/kg/d; n=10) and Control gruoup (NF,HF group, n=10) one time daily for16weeks. The role of PARP-1/iNOS was being analyzed in vivo on related genes and protein in the development of T2DM.ResultsPart I:We found good association between Ox-LDL and MS-1apoptosis. Ox-LDL potently diminished the number of viable MS-1cells. Apoptosis was significantly higher in MS-1cells cultured in100or200μg/ml Ox-LDL than in normal medium, with no significant difference between100and200μg/ml Ox-LDL. The suppressive effect by GLP-1on iNOS protein expression in ox-LDL-treated MS-1cells was mediated by PARP-1. GLP-1(100nmol/L) greatly suppressed the expression of iNOS and the iNOS inhibitor1400W (100μM) had a similar effect. GLP-1was as effective as DPQ and1400W, inhibitors of PARP-1and iNOS, respectively, in inhibiting NO production in MS-1cells. The number of TUNEL-positive cells was significantly increased with Ox-LDL-induced MS-1apoptosis as compared with the control (15.02±0.41%vs.6.32±0.23%, p<0.01). Treatment with GLP-1, DPQ,1400W and Exendin (9-39), antagonist of GLP1-R, markedly reduced the number of TUNEL-positive cells as compared with the control (P<0.05). Part Ⅱ:The PARP-1gene fragments successfully inserted into the pEGM-T-Easy were confirmed by digestion and DNA senquencing;the PARP-1gene fragment inserted into PLVX-AcGFP-N1vector correctly was indentified confirmed by digestion and DNA senquencing;the expression of enhanced green fluoscence protein was observed in MS-1cells transfected with PLVX-AcGFP-Nl under fluorescence microscope. GFP protein was localized in whole cell, while PARP-1is expressed mainly in the cell Plasm. The results of RT-PCR congfirmed that the recombinant vectors expressed PAPR-1mRNA in MS-1cells stably.Part III:Compared with the normal diet group, high fat diet group ApoE-/-mice, total plasma cholesterol ester (TC), triglyceride (TG), low density lipoprotein (LDL-C), high density lipoprotein (HDL-C) level was significantly higher (P<0.01), and fasting blood glucose (FBG) was significantly higher than the normal diet group, and with the extension of feeding time, lipid disorders gradually increased. After GLP-1treatment, fasting blood glucose (FBG),ITT and IPGTT were significantly ameliorate in GLP-1and PARP-1inhibitor group compared with HF group (p<0.01and p<0.05, respectively).PARP-1and iNOS mRNA expression and protein levels of GLP-1group were significantly lower than that of HF group (p<0.01and p<0.05respectively) in islet tissues. GLP-1and DPQ application can reduce the damage of islet function by inhibiting the expression of adhesion molecules VCAM-1, P-selectin to alleviate the islet lymphocyte infiltration,destruction,(P<0.05).Conclusions1. GLP-1can effectively protect MS-1cells against Ox-LDL-induced apoptosis by inactivating the PARP-1/iNOS/NO pathway2. PARP-1gene were cloned successfully.The Lentivirus vector PLVX-AcGFP-N1-PARP-1containing human PARP-1gene was successfully constructed and expressed in MS-1cells stably.3. PARP-1and iNOS might have been involved in the pathways of lipid metabolism in inducing injury of islet countered by GLP-1. GLP-1and PARP-1inhibitor DPQ inhibit PARP-1/iNOS pathway activity and reduction of3-NT generation, thus reducing the a cell glucagon levels while increasing the pancreatic β cell insulingeneration, but also by inhibiting the expression of VCAM-1, P-selectin generated, reducing islet lymphocyte infiltration, thereby ameliorate ApoE-/-mouse islet function under high-fat feeding.SignificanceWe offer an insight that Ox-LDL has dose and time-dependent cytotoxic effect on pancreatic islet microvascular endothelial cells (MS-1). GLP-1treatment, by inhibiting apoptosis, represents a therapeutic tool to improve islet vascularization and, indirectly, islet function. We have also successfully constructed cloning vector containing GFP-PARP-1eukaryotic expression vector; and further study of the fat toxicity ApoE-/-mouse islet mechanism of injury and potential use of this vector intervention strategies. The intervention of the GLP-1and the PAPR-1specific inhibitors, can inhibit the islet microvascular endothelial cell apoptosis and improved islet vascular structure and function, and indirectly improve the islet oxygen, nutrient supply, but also can directly inhibit the islet cells pathway activity PARP-1/iNOS, the reduction of a cell glucagon levels while increasing the generation of islet β-cell insulin, but also can reduce the islet lymphocyte infiltration by inhibiting the expression of VCAM-1, P-selectin generated and structural damage. Pathogenic role for the inhibit PARP-1/iNOS pathway in the disease progression in diabetes and to find protection targets of the pancreatic microcirculation and pancreatic functions, which have important meaning and value.