RESUMO
BACKGROUND: The glucagon-like peptide-1 receptor (GLP-1R) agonist - liraglutide (LIR) - is an insulin secretagogue for the treatment of diabetes and has been proven to have therapeutic potential in the treatment of COPD. Evidence suggested that activating GLP-1R signaling might have immunomodulating and anti-inflammatory effects. COPD is characterized by dysregulation of immunity, oxidative stress, and excessive inflammation responses. The aim of this study was to investigate whether GLP-1R signaling had a regulatory role in COPD immunity. PATIENTS AND METHODS: Fifty-seven COPD patients in a stable condition and 51 age-, sex-, and smoking history-matched non-COPD subjects provided blood samples for isolation of peripheral blood mononuclear cells (PBMCs). GLP-1R expression was measured, and its association with clinical parameters and plasma cytokines was analyzed. T cell function was assessed at baseline and after regulating GLP-1R expression. RESULTS: GLP-1R expression decreased in circulating PBMCs of COPD patients, which was associated with decreased interferon (IFN)-γ expression. Reduced IFN-γ production stimulated by phytohemagglutinin (PHA) and increased programmed cell death protein 1 (PD-1) expression on T cells were observed in COPD patients compared with non-COPD subjects. Treatment with LIR could upregulate the GLP-1R expression, and this was observed to restore the antigen-stimulated IFN-γ production and downregulate PD-1 expression in T cells. CONCLUSION: PBMCs of COPD patients showed defective GLP-1R signaling and functional T-lymphocyte abnormalities that could be rescued by LIR treatment.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1 , Inflamação/metabolismo , Leucócitos Mononucleares/imunologia , Liraglutida/farmacologia , Estresse Oxidativo/imunologia , Doença Pulmonar Obstrutiva Crônica , Citocinas/sangue , Feminino , Perfilação da Expressão Gênica/métodos , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/imunologia , Humanos , Fatores Imunológicos/farmacologia , Interferon gama/genética , Masculino , Pessoa de Meia-Idade , Fragmentos de Peptídeos/genética , Receptor de Morte Celular Programada 1/genética , Doença Pulmonar Obstrutiva Crônica/genética , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/metabolismo , Transdução de Sinais , Fumar/imunologia , Fumar/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
The present study aimed to investigate the mechanism of glucagonlike peptide1 receptor (GLP1R) agonists in the progression of diabetic peripheral neuropathy (DPN) in streptozotocin (STZ)induced diabetic rats, through inflammatory signaling pathways. The DPN rat model was generated by intraperitoneal injection of STZ and then treated with the GLP1R agonist liraglutide or saline for 8 weeks. These animals were randomly divided into 4 groups (10 rats in each): The normal control + saline group, the normal control + liraglutide group, the diabetic + saline (DM) group and the diabetic + liraglutide (DML) group. The nerve conduction velocity (NCV) in the sciatic nerves of the rats was monitored over a period of 8 weeks. Peripheral serum was obtained for the measurement of blood glucose, tumor necrosis factorα (TNFα), interleukin6 (IL6) and IL1ß level. The protein levels of phosphorylated (p) and total extracellular signalregulated kinase, cJun NH2terminal kinases, p38 mitogenactivated protein kinases (MAPK), and nuclear and cytoplasmic nuclear factorκB (NFκB) were measured through western blot analysis. Sciatic nerve mRNA expression levels of proinflammatory chemokines (TNFα, IL6 and IL1ß), chemokines [monocyte chemoattractant protein1 (MCP1)], adhesion molecules [intercellular adhesion molecule 1 (ICAM1)], neurotrophic factors [neuritin, nerve growth factor (NGF) and neuronspecific enolase (NSE)] and NADPH oxidase 4 (NOX4) were evaluated by reverse transcription-quantitative polymerase chain reaction. Subsequent to 8 weeks of treatment with liraglutide, the density of myelin nerve fibers was partially restored in the DML group. The delayed motor NCV and sensory NCV in the DML group were improved. The IOD value of NOX4 staining in the DML group (24.43±9.01) was reduced compared with that in the DM group (56.60±6.91). The levels of TNFα, IL1ß and IL6 in the peripheral serum of the DML group were significantly suppressed compared with those of the DM group. It was also observed that the mRNA expression levels of TNFα, IL6, IL1ß, MCP1, ICAM1 and NOX4 in the sciatic nerve were attenuated in the DML group. The mRNA expression of neuritin and NGF was significantly increased in the DML group compared with that of the DM group; NSE was reduced in the sciatic nerves of the DML group compared with that of the DM group. Additionally, the protein expression of pp38 MAPK and NFκB in the DML group was significantly suppressed. These data demonstrated that GLP1R agonists may prevent nerve dysfunction in the sciatic nerves of diabetic rats via p38 MAPK/NFκB signaling pathways independent of glycemic control. GLP1R agonists may be a useful therapeutic strategy for slowing the progression of DPN.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Neuropatias Diabéticas/tratamento farmacológico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hipoglicemiantes/uso terapêutico , Liraglutida/uso terapêutico , NF-kappa B/imunologia , Nervo Isquiático/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/imunologia , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Experimental/fisiopatologia , Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/imunologia , Neuropatias Diabéticas/fisiopatologia , Receptor do Peptídeo Semelhante ao Glucagon 1/imunologia , Inflamação/complicações , Inflamação/tratamento farmacológico , Inflamação/imunologia , Inflamação/fisiopatologia , Masculino , Ratos , Ratos Sprague-Dawley , Nervo Isquiático/imunologia , Nervo Isquiático/fisiopatologia , Transdução de Sinais/efeitos dos fármacosRESUMO
AIMS/HYPOTHESIS: Glucagon like peptide-1 (GLP-1) enhances glucose-dependent insulin secretion by binding to GLP-1 receptors (GLP1Rs) on pancreatic beta cells. GLP-1 mimetics are used in the clinic for the treatment of type 2 diabetes, but despite their therapeutic success, several clinical effects of GLP-1 remain unexplained at a mechanistic level, particularly in extrapancreatic tissues. The aim of this study was to generate and characterise a monoclonal antagonistic antibody for the GLP1R for use in vivo. METHODS: A naive phage display selection strategy was used to isolate single-chain variable fragments (ScFvs) that bound to GLP1R. The ScFv with the highest affinity, Glp1R0017, was converted into a human IgG1 and characterised further. In vitro antagonistic activity was assessed in a number of assays: a cAMP-based homogenous time-resolved fluorescence assay in GLP1R-overexpressing cell lines, a live cell cAMP imaging assay and an insulin secretion assay in INS-1 832/3 cells. Glp1R0017 was further tested in immunostaining of mouse pancreas, and the ability of Glp1R0017 to block GLP1R in vivo was assessed by both IPGTT and OGTT in C57/Bl6 mice. RESULTS: Antibodies to GLP1R were selected from naive antibody phage display libraries. The monoclonal antibody Glp1R0017 antagonised mouse, human, rat, cynomolgus monkey and dog GLP1R. This antagonistic activity was specific to GLP1R; no antagonistic activity was found in cells overexpressing the glucose-dependent insulinotropic peptide receptor (GIPR), glucagon like peptide-2 receptor or glucagon receptor. GLP-1-stimulated cAMP and insulin secretion was attenuated in INS-1 832/3 cells by Glp1R0017 incubation. Immunostaining of mouse pancreas tissue with Glp1R0017 showed specific staining in the islets of Langerhans, which was absent in Glp1r knockout tissue. In vivo, Glp1R0017 reversed the glucose-lowering effect of liraglutide during IPGTTs, and reduced glucose tolerance by blocking endogenous GLP-1 action in OGTTs. CONCLUSIONS/INTERPRETATION: Glp1R0017 is a monoclonal antagonistic antibody to the GLP1R that binds to GLP1R on pancreatic beta cells and blocks the actions of GLP-1 in vivo. This antibody holds the potential to be used in investigating the physiological importance of GLP1R signalling in extrapancreatic tissues where cellular targets and signalling pathways activated by GLP-1 are poorly understood.
Assuntos
Anticorpos/imunologia , Receptor do Peptídeo Semelhante ao Glucagon 1/antagonistas & inibidores , Receptor do Peptídeo Semelhante ao Glucagon 1/imunologia , Animais , Células CHO , Cálcio/metabolismo , Linhagem Celular , Cricetulus , AMP Cíclico/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/metabolismo , Incretinas/metabolismo , Insulina/metabolismo , Camundongos , Biblioteca de PeptídeosRESUMO
INTRODUCTION: Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are injectable agents used for the treatment of hyperglycemia in type 2 diabetes. The interest for this pharmacological class is rising with the development of once weekly compounds and the demonstration of a potential reduction in cardiorenal outcomes. Areas covered: The paper describes the main pharmacokinetic/pharmacodynamic characteristics of dulaglutide, a new once-weekly GLP-1 RA. Dulaglutide was extensively investigated in the phase-3 AWARD program, which demonstrated its safety and efficacy when compared to placebo or active glucose-lowering agents in patients treated with diet alone, metformin or sulfonylurea monotherapy, oral dual therapies and basal insulin. In both Caucasian and Japanese patients, comparative trials showed better glucose control with dulaglutide, with a minimal risk of hypoglycemia and weight loss, but at the expense of an increased dropout rate due to side effects, mostly transient gastrointestinal disturbances. Dulaglutide proved its non-inferiority versus liraglutide and the safety and tolerance profile is similar to that of other GLP-1 RAs. Expert opinion: The once-weekly formulation and the combined positive effects on both glucose control and weight improves patient satisfaction despite nausea. Dulaglutide must prove its capacity to reduce cardiovascular and diabetic complications in the ongoing prospective REWIND trial.