h-89 and exendin-(9-39)

In type 2 diabetes, lipotoxicity damages islet microvascular endothelial cells (IMECs), leading to pancreatic islet β cell dysfunction directly or indirectly. Glucagon-like peptide-1 (GLP-1) and its analogs have beneficial roles in endothelial cells. However, the protective effects of GLP-1 agents on IMECs and their potential mechanism remained obscure. In this study, exposure of MS-1 (a cell line derived from mouse IMECs) to different concentrations of palmitic acid (PA) was used to establish an injury model. The cells exposed to PA (0.25 mmol/L) were treated with a GLP-1 analog liraglutide (3, 10, 30, and 100 nmol/L). Reactive oxygen species (ROS) generation, apoptosis-related protein level, and endothelin-1 production were detected. The protein levels of signaling molecules were analyzed and specific inhibitors or blockers were used to identify involvement of signaling pathways in the effects of liraglutide. Results showed that PA significantly increased ROS generation and the levels of pro-apoptotic protein Bax, and decreased the levels of anti-apoptotic protein Bcl-2 and the mRNA expression and secretion of endothelin-1. Meanwhile, PA downregulated the protein levels of GLP-1 receptor (GLP-1R), phosphorylated protein kinase A (PKA), guanosine 5'-triphosphate cyclohydrolase 1 (GTPCH1), and endothelial nitric oxide synthase (eNOS). Furthermore, liraglutide ameliorated all these effects of PA in a dose-dependent manner. Importantly, GLP-1R antagonist exendin (9-39), PKA inhibitor H89, GTPCH1 inhibitor 2,4-diamino-6-hydroxypyrimidine, or NOS inhibitor N-nitro-l-arginine-methyl ester abolished the liraglutide-mediated amelioration in PA-impaired MS-1 cells. In conclusion, liraglutide ameliorates the PA-induced oxidative stress, apoptosis, and endothelin-1 secretion dysfunction in mouse IMECs through GLP-1R/PKA and GTPCH1/eNOS signaling pathways.

Topics: Animals; bcl-2-Associated X Protein; Cyclic AMP-Dependent Protein Kinases; Endothelin-1; Endothelium, Vascular; Glucagon-Like Peptide-1 Receptor; GTP Cyclohydrolase; Islets of Langerhans; Isoquinolines; Liraglutide; Mice; Nitric Oxide Synthase Type III; Oxidative Stress; Palmitates; Peptide Fragments; Protective Agents; Reactive Oxygen Species; Signal Transduction; Sulfonamides

Hyperglycemia-induced cardiomyocyte apoptosis contributes to diabetic cardiomyopathy. Glucagon-like peptide-1 (Glp1) receptor (Glp1r) agonists improve cardiac function and survival in response to ischemia-reperfusion and myocardial infarction. The present studies assessed whether Glp1r activation exerts direct cardioprotective effects in response to hyperglycemia. Treatment with the Glp1r agonist Exendin-4 attenuated apoptosis in neonatal rat ventricular cardiomyocytes cultured in high (33 mM) glucose. This protective effect was mimicked by the cAMP inducer forskolin. The Exendin-4 protective effect was blocked by the Glp1r antagonist Exendin(9-39) or the PKA antagonist H-89. Exendin-4 also protected cardiomyocytes from hydrogen peroxide (H2O2)-induced cell death. Cardiomyocyte protection by Exendin-4 was not due to reduced reactive oxygen species levels. Instead, Exendin-4 treatment reduced endoplasmic reticulum (ER) stress, demonstrated by decreased expression of glucose-regulated protein-78 (GRP78) and CCAT/enhancer-binding homologous protein (CHOP). Reduced ER stress was not due to activation of the unfolded protein response, indicating that Exendin-4 directly prevents ER stress. Exendin-4 treatment selectively protected cardiomyocytes from thapsigargin- but not tunicamycin-induced death. This suggests that Exendin-4 attenuates thapsigargin-mediated inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase-2a (SERCA2a). High glucose attenuates SERCA2a function by reducing SERCA2a mRNA and protein levels, but Exendin-4 treatment prevented this reduction. Exendin-4 treatment also enhanced phosphorylation of the SERCA2a regulator phospholamban (PLN), which would be expected to stimulate SERCA2a activity. In sum, Glp1r activation attenuates high glucose-induced cardiomyocyte apoptosis in association with decreased ER stress and markers of enhanced SERCA2a activity. These findings identify a novel mechanism whereby Glp1-based therapies could be used as treatments for diabetic cardiomyopathy.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cells, Cultured; Colforsin; Diabetic Cardiomyopathies; Endoplasmic Reticulum Stress; Enzyme Activation; Exenatide; Glucagon-Like Peptide-1 Receptor; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Hyperglycemia; Hypoglycemic Agents; Isoquinolines; Membrane Proteins; Myocytes, Cardiac; Oxidative Stress; Peptide Fragments; Peptides; Phosphorylation; Protein Kinase Inhibitors; Rats; Receptors, Glucagon; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Transcription Factor CHOP; Tunicamycin; Unfolded Protein Response; Venoms

The aim of this project is to establish a GLP-1 signaling pathway targeted cell model, for screening the new class of GLP-1 receptor agonists as anti-diabetic candidates. Firstly construct a recombined plasmid with multi-copied specific response element (RIP-CRE) regulated by GLP-1 signaling pathway and E-GFP reporter gene. Transient transfect this recombined plasmid into islet cell NIT-1, then detect the responsibility of transfected cell to GLP-1 analogue, Exendin 4. For secondly, use stable transfection and monocloning cell culture to obtain a GLP-1 signaling-specific cell line. It indicates that this cell model can response to Exendin 4, which response can be completely inhibited by GLP-1 receptor antagonist, Exendin 9-39, further showing GLP-1 receptor specific activity with a cAMP-PKA-independently mechanism. Establishment of this novel cell model can be used in high-throughput drug screening of peptides or small molecular GLP-1 analogues.

Topics: Animals; Cell Line; Cyclic AMP Response Element Modulator; Cyclic AMP-Dependent Protein Kinases; Drug Delivery Systems; Drug Evaluation, Preclinical; Exenatide; Genes, Reporter; Glucagon-Like Peptide-1 Receptor; Green Fluorescent Proteins; Hypoglycemic Agents; Islets of Langerhans; Isoquinolines; Peptide Fragments; Peptides; Plasmids; Rats; Receptors, Glucagon; Recombinant Proteins; Signal Transduction; Sulfonamides; Transfection; Venoms

Glucagon-like peptide-1 (GLP-1) is a proglucagon-derived hormone with cellular protective actions. We hypothesized that GLP-1 would protect the endothelium from injury during inflammation. Our aims were to determine the: (1) effect of GLP-1 on basal microvascular permeability, (2) effect of GLP-1 on increased microvascular permeability induced by lipopolysaccaride (LPS), (3) involvement of the GLP-1 receptor in GLP-1 activity, and (4) involvement of the cAMP/PKA pathway in GLP-1 activity. Microvascular permeability (L(p)) of rat mesenteric post-capillary venules was measured in vivo. First, the effect of GLP-1 on basal L(p) was measured. Second, after systemic LPS injection, L(p) was measured after subsequent perfusion with GLP-1. Thirdly, L(p) was measured after LPS injection and perfusion with GLP-1+GLP-1 receptor antagonist. Lastly, L(p) was measured after LPS injection and perfusion with GLP-1+inhibitors of the cAMP/PKA pathway. Results are presented as mean area under the curve (AUC)+/-SEM. GLP-1 had no effect on L(p) (AUC: baseline=27+/-1.4, GLP-1=1+/-0.4, p=0.08). LPS increased L(p) two-fold (AUC: LPS=54+/-1.7, p<0.0001). GLP-1 reduced the LPS increase in L(p) by 75% (AUC: LPS+GLP-1=34+/-1.5, p<0.0001). GLP-1 antagonism reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+antagonist=46+/-2.0, p<0.001). The cAMP synthesis inhibitor reduced the effects of GLP-1 by 60% (AUC: LPS+GLP-1+cAMP inhibitor=46+/-1.5, p<0.0001). The PKA inhibitor reduced the effects of GLP-1 by 100% (AUC: LPS+GLP-1+PKA inhibitor=56+/-1.5, p<0.0001). GLP-1 attenuates the increase in microvascular permeability induced by LPS. GLP-1 may protect the endothelium during inflammation, thus decreasing third-space fluid loss.

Topics: Animals; Capillary Permeability; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dideoxyadenosine; Endothelium, Vascular; Enzyme Inhibitors; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Inflammation; Isoquinolines; Lipopolysaccharides; Mesentery; Peptide Fragments; Perfusion; Protein Kinase Inhibitors; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Rolipram; Sulfonamides; Venules