Effects Of Hypothalamic ARC PYY_1-36 On The Feeding,Gastric Motility And Energy Metabolism In Rats And Its Potential Mechanism

Objective:Recent studies on obesity have focused on the field of neuroendocrine regulation of energy balance.It is known that brain gut peptide plays a key role in the regulation of body weight and energy metabolism balance,and casein peptide（PYY）is a brain gut peptide,which is a more effective anorectic peptide and plays an important role in the etiology of obesity.There are two forms of PYY in the circulation of the body,namely PYY_1-36 and PYY_3-36.PYY_1-36 is cleaved by the specific cell surface enzyme dipeptidyl peptidase-IV（DPPIV）to form PYY_3-36.At present,little is known about the physiological function regulation of PYY_1-36,especially the central effect and mechanism of PYY_1-36 on feeding or energy metabolism research.ARC is one of the nuclei involved in feeding,gastric motility,gastric acid secretion and other important regulation energy balance.Therefore,it is necessary to explore the regulation of ARC PYY_1-36 on energy balance.Therefore,the purpose of this experiment was to investigate the effects and potential mechanisms of PYY_1-36 on feeding,gastric motility,and energy metabolism in rats.Methods:1.The expression of PYY receptor 2（Y2R）in the arcuate nucleus（ARC）of rat hypothalamus was observed by fluorescent immunohistochemical staining 2.To observe the expression of Y2R mRNA in the hypothalamic ARC of rats by RT PCR;3.To observe the expression of Y2R protein in the hypothalamic ARC of rats by western Blot; 4.ARC microinjection of PYY_1-36,observe whether ARC has PYY_1-36 sensitive neurons (PYY_1-36-N):30 rats were randomly selected.PYY_1-36 or Y2 receptor antagonist SF-11 was injected into rat ARC through glass microelectrode to observe the change of neuron firing frequency in ARC to identify PYY sensitive neurons;5.Microinjection of PYY_1-36 or SF-11 into the ARC,observed effects on feeding and energy metabolism in rats:24 rats in ARC were randomly divided into 4 groups（n=6）: NS group（0.5μl NS）,PYY1-36 group(0.5 nmol/0.5μL PYY_1-36),SF-11 group（1 nmol/0.5μL SF-11）and PYY_1-36+SF-11 group(1 nmol/0.25μL SF-11+0.5 nmol/0.25 μL PYY_1-36).Through the comprehensive experimental animal monitoring system （CLAMS）,the effects of ARC microinjection of PYY_1-36 on food and water uptake, oxygen consumption(V_O2),CO₂ production(V_CO2),and heat production（Heat）in rats for 4 h day and night were observed; 6.Effect of ARC microinjection of PYY_1-36 on gastric motility in rats:24 rats in ARC were randomly divided into 4 groups（n=6）:NS group（0.5μl NS）,PYY_1-36 group(0.5 nmol/0.5μL PYY_1-36),SF-11 group（1 nmol/0.5μL SF-11）and PYY_1-36+SF-11 group (1 nmol/0.25μL SF-11+0.5 nmol/0.25μL PYY_1-36).After 7 days of recovery by ARCcatheterization and gastric placement of random stressors.The drug was injected into the rat ARC through a cannula,and the effect on the amplitude of the gastric contraction and the frequency of contraction was observed.The gastric contraction signal is transmitted from the stressor to the gastrointestinal motility transducer,where it is converted into an electrical signal input computer,and the gastrointestinal motion data is analyzed by Powerlab multi-channel biological signal acquisition and processing system.;Results:1.Immunofluorescence histochemical experiments showed that Y2 receptor immunopositive cells were distributed in the rat AII of hypothalamus; 2.RT-PCR and Wethern Blot results showed that Y2R mRNA and protein were expressed in rat adipose ARC,further confirming the presence of Y2R receptor in rat hypothalamic ARC; 3.Single-cell discharge records showed that 43 neurons were recorded in the hypothalamic ARC,and 37（37/43,86.05%）neurons responded to PYY_1-36,exhibiting changes in discharge activity,and defined these neurons as PYY_1-36 reactive neurons （PYY-N）,the remaining 6 neurons did not change significantly after the administration of PYY_1-36.Among the 37 PYY-N,21（21/37,56.76%）neurons showed an increase in the frequency of discharge of PYY_1-36（P<0.05）,with an average increase of 90.47±12.33%.These neurons were called PYY_1-36 excitatory neurons(PYY_1-36-E);the remaining 16（16/37,43.24%）neurons were PYY_1-36 inhibitory neurons(PYY_1-36-I),administration of PYY_1-36 into the ARC,the frequency of neuronal firing was significantly reduced（P<0.001）with an average reduction of 39.20%.If pre-injection SF-11 into the ARC,the excitatory or inhibitory effect of PYY_1-36 on PYY-N could bepartially blocked（P<0.05）.However,administration of SF-11 alone had no significant effect on PYY-N discharge activity（P>0.05）.The results suggest that there are PYY_1-36 reactive neurons in ARC,and PYY_1-36 can regulate the excitatory properties of neurons by activating Y2 receptors; 4.The effect of ARC microinjection of PYY_1-36 on feeding and drinking water in ratsshowed that,compared with NS group,microinjection of PYY_1-36 into the ARC,the food intake per hour at 0-4 h significantly decreased（P<0.01）.Microinjection of SF-11 into the ARC,the food intake per hour at 0-4 h significantly increased（P<0.05）;compared with PYY_1-36,microinjection of SF-11+PYY_1-36 mixture,the food intake per hour at 0-4 h significantly increased（P<0.05）.It is suggested that both internal andexternal PYY_1-36 can inhibit the feeding effect of rats,and the PYY_1-36 antifeedant effect can be partially blocked by Y2 receptor（Y2R）antagonist,that is,the inhibition of PYY_1-36 on food intake may be Realized by the Y2R signal path.Compared with the NS group,Microinjection of PYY_1-36 or SF-11into the ARC showed no significant change in the cumulative water intake of the rats at 0-4 h（P>0.05）.Compared with the PYY_1-36 group,microinjection of SF-11+PYY_1-36 mixture,there was no significant difference in the cumulative water intake of rats 0-4 h（P>0.05）.It is suggested thatPYY_1-36 may have no significant regulatory effect on water uptake in rats; 5.The effect of ARC microinjection of PYY_1-36 on energy metabolism in rats showed that compared with NS group,Microinjection of PYY_1-36 into the ARC,significantly increased V_O2 in rats（P<0.05）,accompanied by significant V_CO2 and caloric production.Increased（P<0.05）;compared with NS group,ARC injection of SF-11,rat V_O2 significantly decreased（P<0.05）,accompanied by a significant decrease in V_CO2 and caloric production（P<0.05）;Compared with the PYY_1-36 group,microinjection of Y2Rantagonist SF-11+PYY_1-36 mixture into the ARC,the promotion of V_O2,V_CO2 or caloric production by PYY_1-36 was partially attenuated（P<0.05）.It is suggested that both endogenous and exogenous PYY_1-36 of ARC are involved in the regulation of energy metabolism in rats.The negative balance regulation effect of PYY_1-36 energy metabolism can be partially blocked by Y2 receptor antagonists,that is,the increase of energy metabolism of PYY_1-36 may be partially achieved through the Y2R signaling pathway; 6.The effect of ARC microinjection of PYY_1-36 on gastric motility in rats showed that ARC microinjection of PYY_1-36 significantly decreased the gastric contraction amplitude（P<0.01）and contraction frequency（P<0.01）after 5 min.ARC microinjection of SF-11 showed a significant increase in gastric contraction amplitude （P<0.01）and contraction frequency（P<0.01）after 5 min.If ARC was injected with SF-11+PYY_1-36 mixture,the gastric motility effect of PYY_1-36 was partially blocked（P<0.05）.It is suggested that PYY_1-36 may be involved in the regulation of gastric motility in rats through the Y2R signaling system.Conclusions:ARC has PYY action sites;Both internal and external PYY are involved in the regulation of feeding,gastric motility and energy balance in rats.This effect is partly regulated by the Y2 receptor signaling pathway,and PYY_1-36 is expected to be a novel metabolic regulatory peptide.