| Objective: Orexin-A is one of the important brain-gut peptides secreted by hypothalamus and involved in food intake,which is closely related to the occurrence and development of obesity.The aim of this study is to investigate the effects of orexin-A on firing activity of gastric distension-sensitive(GD)neurons in the basomedial amygdala(BMA)and food intake among the normal rats,diet-induced obesity(DIO)rats and diet-induced resistant(DR)rats and the potential regulation mechanism.Methods: Healthy male Wistar rats were selected,a diet-induced obesity(DIO)and diet-induced resistant(DR)rat model were induced by high-fat diet.The effects of orexin-A and ?-opioid receptor antagonist naloxone on the activity of BMA GD neurons among the normal rats,DIO rats,and DR rats were observed by extracellular recording of neuronal discharge;The effects of orexin-A and naloxone on food intake among the normal rats,DIO rats and DR rats and their potential mechanisms were compared and analyzed by nucleus catheterization and microinjection of drugs;The distribution of orexin-1 receptor(OX-1R)and ?-opioid receptor immunopositive cells in the BMA among the normal rats,DIO rats and DR rats was observed by immunohistochemical staining;The m RNA expression and protein expression levels of orexin-1 receptor(OX-1R)and ?-opioid receptor among the normal rats,DIO rats,and DR rats were detected by Real-time PCR and Elisa.Results:(1)After BMA microinjection of orexin-A,the discharge frequency of BMA GD-sensitive neurons among the normal rats,DIO rats and DR rats increased significantly(P<0.01).This effect was completely blocked by OX-1R receptor antagonist SB-334867 and partly blocked by naloxone.Compared with the normal rats,orexin-A significantly enhanced the discharge-promoting effect of GD-E(P<0.05)or GD-I(P<0.05)neurons in the BMA in DIO rats and DR rats,but there was no significant difference between DIO rats and DR rats(P>0.05);Naloxone significantly inhibited the discharge frequency of GD-E and GD-I neurons in the BMA of above three groups of rats,and compared with the normal rats,naloxone significantly inhibited GD-E and GD-I neurons in DIO rats(P<0.05),and had stronger inhibitory effect on GD-E and GD-I neurons in DR rats than in DIO rats(P < 0.05).The results suggest that orexin-A may be involved in the regulation of gastric afferent information,which may be mediated by OX-1R signaling pathway,and the signal of ?-opioid receptor may also be involved in this process.(2)BMA microinjection of orexin-A could significantly increase the 0-2 h food intake of the normal rats,DIO rats and DR rats(P<0.01),but had no significant effect on the 2-4 h food intake(P>0.05).Compared with the normal rats,BMA injected orexin-A significantly increased the food intake of DIO rats and DR rats at 0-2h(P<0.05),but there was no significant difference between DIO rats and DR rats(P>0.05).Preadministration of SB-334867 could block feeding-promoting effect of orexin-A(P<0.05).BMA microinjection of naloxone could significantly inhibit the 0-2 h food intake of rats(P<0.05),and the effect was significantly stronger in DR rats than in DIO rats(P<0.05).Further studies showed that naloxone partially blocked orexin-A-induced feeding in rats(P<0.05).These results suggest that orexin-A may be involved in the regulation of feeding through OX-1R signaling pathway,and the signal of ?-opioid receptor may also be involved in this process.(3)Fluorescence immunohistochemical study showed that the expression of OX-1R immunopositive cells in the BMA of DIO rats and DR rats increased significantly compared with the normal rats(P<0.05),but there was no significant difference between DIO rats and DR rats(P>0.05).The expression of ?-opioid receptor immunopositive cells in the BMA of DIO rats and DR rats was also significantly higher than that in the normal rats(P<0.05),and the expression of ?-opioid receptor immunopositive cells in the BMA of DR rats was significantly higher than that in DIO rats(P<0.05).It suggests that high-fat diet may affect rat feeding by inducing changes in orexins receptor expression in the BMA.(4)Real-time PCR results showed that the m RNA expression of BMA OX-1R in DIO rats and DR rats was significantly higher than that in the normal rats(P<0.05),but there was no significant difference between DIO rats and DR rats(P>0.05);the m RNA expression of ?-opioid receptor in DR rats was significantly higher than that in DIO rats(P<0.05),and the m RNA expression of ?-opioid receptor in DIO rats or DR rats was significantly higher than that in the normal rats(P<0.05).It suggests that OX-1R or ?-opioid receptor signaling pathway may be involved in the differential regulation of BMA neurons excitability and feeding in these three groups.(5)ELISA results showed that the protein expression levels of BMA OX-1R in DIO rats and DR rats was significantly higher than that in the normal rats(P<0.05),but there was no significant difference between DIO rats and DR rats(P>0.05);the protein expression levels of ?-opioid receptor in the BMA from high to low was DR rats,DIO rats and normal rats(P<0.05).These results further confirm that the differences in BMA neurons excitability and feeding in these three groups may be related to the changes in the expression of OX-1R receptor or ?-opioid receptor in the central nervous system.Conclusion: BMA orexin-A participates in gastric distension afferent information and feeding regulation in the normal,obese and obese resistant rats through OX-1R signaling pathway,and ?-opioid receptor signaling pathway may also participate in this process.The similarities and differences in orexin-A regulation of food intake in the normal,obese and obese resistant rats may be related to the expression of OX-1R or ?-opioid receptor. |
PDF Full Text Request