| Objective Obesity is currently one of the most serious health problems in the world.Excessive food intake is a major cause of obesity.Therefore,in-depth research on the regulatory mechanism of food intake is helpful to find targets and treat obesity.Feeding function is mainly regulated by central nervous system and intestinal endocrine system.Previous studies have shown that calcium-sensitive receptor(CaSR)is widely distributed in brain regions related to central and feeding regulation,including hypothalamus,brainstem and limbic system,especially in the area postrema(AP).There are close fibrous connections between AP and solitary tract nuclei(NTS),and glucagon-like peptide-1(GLP-1)positive neurons in NTS have single synaptic connections with parabrachial nuclei(PBN).Therefore,this study mainly explored the mechanism by which CaSR in AP affects feeding through the NTS-PBN GLP-1pathway.Method1.The influence of CaSR agonist-R568 administered in the AP on 0-24 h food intake of normal mice and obese mice was observed by feeding behavior analysis;The effects of GABA-A receptor blockers and GLP-1R blockers on the inhibition of feeding induced by R568;2.Enzyme linked immunosorbent assay(ELISA)was used to observe the effects of microinjection of R568 in the AP on the content of neurotransmitter γ-aminobutyric acid(GABA),GLP-1 in the NTS,and the changes of the levels of CaSR in the AP,GLP-1 in the NTS and GLP-1R in the PBN of normal and obese mice.3.Anterograde virus(rAAV-hSyn-mCherry-WPRE-hGH pA)was injected into the AP,and the nerve fiber projection of AP-NTS was observed and combined withGLP-1 immunofluorescence staining;4.GLP-1 neurons in Gcg-IRES-CreERT2 mice were inhibited by chemogenetic method(Microinjected with AAV-EFla-DIO-h M4D(Gi)-EGFP-WPRE into the NTS),and the effect on feeding inhibition induced by R568 was observed;5.Immunofluorescence staining(IF)was used to observe the co-staining of GLP-1and c-fos immunopositive cells in the NTS and the co-staining of glucagon-like peptide-1 receptor(GLP-1R)and c-fos in the PBN after microinjection of R568 into the AP;6.A high-fat diet-induced obese mouse model was established,after 2 weeks of continuous microinjection of R568 into the AP of obese mice,the changes of body weight were observed.Result1.The feeding results showed that compared with DMSO group,after microinjection of R568 into the AP,the food intake of mice decreased significantly within 0-1h and1-2h(P<0.05).2.ELISA results showed that compared with the control group,GABA content in the AP increased significantly after microinjection of R568(P<0.05),the release of GLP-1 in NTS was also significantly increased(P<0.05).3.Immunofluorescence staining results showed that the number of GLP-1 and c-fos immunoreactive neurons in the NTS increased significantly after microinjection of R568 in the AP compared with the control group,the co-expression number of GLP-1and c-fos immunoreactive neurons also increased significantly(P<0.05).4.The anorexia effect of R568 was weakened by microinjection of GABA-A receptor blocker-Bicuculline into the AP(P<0.05).5.The results of anterograde tracking technology combined with immunofluorescence staining showed that after microinjection of anterograde virus into the AP,obvious anterograde fibers were observed in the NTS under fluorescence microscope;IF results showed many anterograde fibers around GLP-1 positive neurons.This indicates that AP neurons project to NTS GLP-1 positive neurons.6.Elisa results showed that microinjection of GABA-A receptor blocker Bicuculline in the AP attenuated the increase of GLP-1 content induced by R568(P<0.05).7.Chemogenetic results showed that inhibition of GLP-1 neurons of NTS reduced the anorexia effect induced by R568(P<0.05).8.IF showed that the number of GLP-1R and c-fos immunopositive cells in the PBN increased significantly after microinjection of R568 into the AP compared with the control group(P<0.05).9.Pre-microinjection of GLP-1R blocker Exendin(9-39)into the PBN inhibited the anorexia effects induced by R568(P<0.05).10.The results of feeding showed that,compared with the control group,the food intake of obese mice was significantly reduced within 0-1h and 1-2h after microinjection of R568 into the AP(P<0.05).11.The results showed that the content of CaSR in obese mice was significantly increased,and the levels of GLP-1 in the NTS and GLP-1R in the PBN were also significantly decreased(P<0.05);IF results also showed that the number of CaSRpositive neurons in obese mice increased significantly,while the number of GLP-1positive neurons in the NTS and GLP-1R in the PBN decreased significantly.12.ELISA results showed that the weight of obese mice decreased significantly after14 days of microinjection of R568 into the AP compared with DMSO group.GLP-1content in the NTS was significantly increased(P<0.05),the content of GLP-1R in the PBN was also significantly increased(P<0.05).The expression of GLP-1 positive neurons in the NTS and GLP-1R in the PBN was increased by IF.ConclusionIn normal mice,activation of CaSR in AP can promote the release of GABA,thereby causing a short-term decrease in food intake.This effect is related to the activation of the AP-NTS-PBN GLP-1 pathway,but the GABA neurons of AP are not involved in this effect.The inhibition of NTS-PBN GLP-1 pathway in obese mice may be related to the occurrence of obesity;The expression of CaSR in AP of obese mice significantly increased and 2-week chronic administration of R568 into AP can increase the excitability of the AP-NTS-PBN GLP-1 pathway by activating CaSR,thereby inhibiting feeding and reducing mouse weight.Our research will provide new targets for the treatment of obesity and new directions for the study of energy metabolism diseases. |
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