| Objective:With the economic development of modern society and the improvement of people’s living standards,obesity has become a common disorder of energy metabolism and nutrition in modern society.In recent years,the incidence of obesity is increasing year by year in China.Obesity can easily cause a variety of complications(such as diabetes,cardiovascular and cerebrovascular diseases,hyperlipidemia and cancer,etc.),promote aging and death.As the pathogenesis of obesity is very complex,obesity and its metabolic disorder has become a research hotspot.Neuropeptide Y(NPY)is a single-chain polypeptide composed of 36 amino acids,which is widely distributed in the central and peripheral nervous system.NPY has a variety of biological activities,especially plays an important role in the regulation of energy balance.In the center,NPY is mainly distributed in the hypothalamic arcuate nucleus(ARC),ARC is located at the bottom of the third ventricle,adjacent to the median eminence of the periventricular organ,mainly involved in the regulation of food intake,cardiovascular activity,endocrine and other regulation,but also plays an important role in the regulation of gastrointestinal function.However,it has not been reported whether NPY is involved in the regulation of gastric motility and feeding in hypothalamic ARC,especially in the process of obesity.In this study,molecular biology,fluorescence immunohistochemical staining,radioligand receptor binding study,single cell extracellular discharge and in vivo gastric motility recording were used to observe the effects of microinjection of NPY on the discharge activity of gastric afferent information neurons received by hypothalamic ARC,as well as the effects on gastric motility,gastric emptying and food intake in rats and its potential mechanism,and to analyze and compare the differences of these effects between normal and obese rats.The aim is to improve the central mechanism of obesity and to provide new research ideas for the prevention and treatment of energy metabolism-related diseases such as obesity.Methods:(1)Healthy male 5 week old SD rats,were fed with 45% fat-supplied high-fat diet for 6weeks to establish a nutritional obesity model.The body weight of the rats was observed regularly every week,and 20% of the body weight of the rats fed with more than 20% of the body weight was regarded as Diet Induced Obesity(DIO)rats.(2)Using single extracellular discharge recording,the effects of hypothalamic ARC microinjection of NPY and NPY-1R antagonist GR231118 on the activity of gastric distention sensitive neurons(GD)in hypothalamic ARC of normal and DIO rats were observed and compared.(3)The effects of hypothalamic ARC microinjection of NPY on gastric motility,gastric emptying and food intake(standard,high fat,high sugar and high protein diet)in normal and DIO rats were observed and compared by nuclear catheterization and nuclear microinjection of drugs.(4)The expressions of ARC NPY and NPY-1R proteins and immunoreactive cells in hypothalamus of normal and DIO rats were observed by Western Blot and fluorescence immunohistochemical staining.(5)The binding capacity of ARC NPY ligand and NPY-1 receptor in hypothalamus of normal and DIO rats was analyzed by radioligand receptor binding assay.Results:(1)Effect of NPY on discharge activity of gastric distention(GD)sensitive neurons in hypothalamic ARC.This study found that GD neurons,including gastric distention inhibitory(GD-I)neurons and gastric distention excitatory(GD-E)neurons,were distributed in hypothalamic ARC.In normal and DIO rats,microinjection of NPY into hypothalamic ARC excited most(normal rats,61.70%: obese rats,67.57%)GD-I neurons(P<0.05),while most GD-E neurons(normal rats,60.81%: obese rats,69.13%)were inhibited(P<0.05).If NPY-1R antagonist GR231118 was injected in advance,the inhibitory or excitatory effect of NPY on hypothalamic ARC GD neurons could be completely blocked(P<0.05).In Diet Induced Obesity rats,the inhibitory/excitatory effect of NPY on GD neurons(GD-E/GD-I)was significantly stronger than that of normal rats(P<0.05).(2)The effects of hypothalamic ARC microinjection of NPY on gastric motility in rats in vivo were studied.The results showed that after hypothalamic ARC microinjection of NPY the amplitude and frequency of gastric motility in normal and Diet Induced Obesity rats increased significantly(P<0.05),and the amplitude and frequency of gastric motility induced by NPY could be completely blocked by ARC microinjection of NPY+GR231118 mixture(P<0.05).Gastric motility index(MI%)analysis showed that MI% increased significantly in both normal and Diet Induced Obesity rats.Although there was no significant difference in gastric motility index(MI%)between Diet Induced Obesity rats and normal rats,the increase rate of MI% in obese rats was more significant than that in normal rats(P<0.05).(3)The effect of hypothalamic microinjection of NPY into ARC on gastric emptying in rats.The results showed that NPY microinject into hypothalamus can significantly increased gastric emptying in normal and Diet Induced Obesity rats(P<0.05),NPY-1R antagonist GR231118 could completely block the promoting effect of NPY on gastric emptying in normal and Diet Induced Obesity rats(P<0.05),and the promoting effect of NPY on gastric emptying in Diet Induced Obesity rats was more significant than that in normal rats(P<0.05).(4)Effects of hypothalamic ARC microinjection of NPY on different feed intake in rats in the gastric motility experiment,it was found that ARC NPY also had a significant effect on food intake in rats.Further study showed that the intake of standard diet or high fat diet of hypothalamic ARC microinjection NPY,rats increased significantly from 0 to 2 h(P<0.05),but there was no significant change in 2-4 h food intake(P>0.05).And there was no significant change in the intake of high sugar diet or high protein diet in hypothalamic ARC microinjection NPY(P>0.05).Microinjection of NPY+GR231118 mixture into ARC could completely block the food-promoting effect of NPY on rats(P<0.05),and the food intake of obese rats at 0-2 h was significantly higher than that of normal rats(P<0.05),and the promoting effect of hypothalamic ARC microinjection of NPY on food intake of obese rats was more significant(P<0.05).(5)Fluorescence immunohistochemical study on the expression of ARC NPY and NPY-1R immunoreactive cells in hypothalamus showed that NPY and its receptor NPY-1R immunoreactive cells were expressed in hypothalamic ARC in normal and Diet Induced Obesity rats(P<0.05),and the expression of NPY and NPY-1R immunoreactive cells in ARC of Diet Induced Obesity rats was significantly higher than that of normal rats(P<0.05).(6)Expression of ARC NPY and NPY-1R protein in hypothalamus Western Blot study showed that NPY and NPY-1R protein were expressed in hypothalamus ARC of normal and Diet Induced Obesity rats(P<0.05),and the expression of NPY or NPY-1R protein in hypothalamus ARC of Diet Induced Obesity rats was significantly higher than that of normal rats(P<0.05).(7)Study on the binding activity of ARC NPY ligand and NPY-1 receptor in hypothalamus in Diet Induced Obesity rats,the specific binding activity of NPY-1R to ligand NPY in hypothalamic ARC of Diet Induced Obesity rats was significantly higher than that of normal rats(P<0.05).Conclusion:There are neurons in the hypothalamic ARC that receive sensory afferent information from the stomach and respond to NPY.NPY participates in the regulation of GD neuronal excitability,gastric motility,gastric emptying and selective feeding through NPY-1 receptor pathway,and this regulation effect is more significant in Diet Induced Obesity rats,which may be related to the increased expression of NPY or NPY-1 receptors in ARC of obese rats,and may also be related to the increased binding of NPY and NPY-1R in ARC of obese rats. |
