| Gonadotropin-inhibitory hormone(Gn IH)is the first identified avian RFamide peptide that acts directly on the pituitary to inhibit the release of gonadotropin from the hypothalamus of quail.After the discovery of Gn IH in birds,RFamide-related peptide-3(RFRP-3,the orthologs of Gn IH)has been further identified in a number of mammals.Gn IH/RFRP-3 has been established as playing an important role in regulating various biologic functions via its G protein-coupled receptor 147(GPR147),such as reproduction,immunity and stress.With the deeper research of RFRP-3,the regulatory role of neuropeptide in energy metabolism has been gradually revealed,but its mechanism is still unclear.Therefore,in this study,SD rats were treated with acute or chronic intraperitoneal injection of different doses of RFRP-3 to explore the effects of the neuropeptide on feeding behavior,glycolipid metabolism,islet morphology and insulin resistance.The results will provide a theoretical basis for RFRP-3 to participate in the research of energy mechanism.The specific methods and results are as follows:Experiment 1,Sprague-Dawley rats were divided into 3 groups randomly.Rats(n=10 per group,half male and half female)received chronic intraperitoneal injections of different doses of RFRP-3(0,1 and 10 μg/100 μL,each time 200 μL of RFRP-3 dissolved in a 0.9% saline solution)for 14 days,the aim of this trial was to investigate the effects of RFRP-3 in changing feeding behavior and lipid metabolism.Microstructural analyses of meal pattern showed that the effect of RFRP-3 on increasing appetite in rats was related to the increase of meal frequency and meal time,the decrease of meal quantity,meal rate,meal time and the meal interval.In terms of body mass and lipid metabolism,RFRP-3 significantly increased the body weights and Lee index.Feed conversion rate was improved.Serum total triglyceride and cholesterol levels were significantly increased.Interestingly,the level of NPY m RNA expression was markedly increased but the level of POMC m RNA expression was declined.Experiment 2,we investigated whether RFRP-3 involved in the regulation of glucose homeostasis and glucometabolism-related hormones concentration in rats in vivo(Grouping and injection method were the same as experiment 1).The results showed that fasting blood glucose levels were dramatically increased in a dose-depended manner at RFRP-3 injected 30 min;Notably,the glucagon and epinephrine were significantly increased,whereas growth hormone was significantly decreased at 15 min post injection;Furthermore,the effect of RFRP-3 inhibited the elimination of exogenous glucose and significantly inhibited the secretion of glucose-stimulated insulin in rats.Experiment 3,In this study,the distribution of RFRP-3 and GPR147 in rat pancreas were analyzed at the morphological and molecular levels.Furthermore,the aim of this trial was to study the effects of intraperitoneal injection of RFRP-3(Grouping and injection method were the same as experiment 1)on the synthesis and secretion of pancreatic islet cytokines and their morphology,which lay a theoretical basis for further research on the physiological functions of RFRP-3.Notably,we confirmed that RFPR-3 and GPR147 were both expressed at m RNA and protein levels in the pancreas.In addition,immunofluorescence double straining data revealed that RFRP-3 was mainly co-localized with glucagon,but GPR147 was primarily co-localized with glucagon and insulin in the pancreatic islet.Interestingly,the level of glucagon protein expression was markedly increased however the level of insulin protein expression was declined in the pancreatic islet.Furthermore,anatomical and histological data documented that chronic intraperitoneal injection of RFRP-3can cause compensatory growth of islet in rats,which is mainly due to the increase of islet α and β-cells.Experiment 4,we attempted to decipher the molecular mechanism underlying the RFRP-3 mediated insulin resistance and inflammation response in insulin-stimulated glucose disposal tissues(Grouping and injection method were same as experiment 1).We confirmed that the expression of RFRP-3m RNA and protein were not investigated in liver,skeletal muscle and white adipose tissue,but the expression of GPR147 m RNA and protein were detected in these tissues.In addition,the results of insulin tolerance test showed that RFRP-3 reduced insulin sensitivity in acute and chronic injection rats.This conclusion was further supported by the reduced expression of insulin receptor and GLUT4,as well as suppression of the GSK-3β signalling in liver and white adipose tissue.Furthermore,our inflammation protein chip array analysis confirmed that the concentration of IL-1α,IL-1β,MCP-1 and TNF-α were significantly increased in serum of the chronic RFRP-3 intraperitoneal injected rats.These results were validated in the high expression of IL-1β and TNF-α in liver and white adipose tissues.As noted above,these changes in decreased uptake of glucose,suppression of insulin signaling and increased inflammation response may be responsible for RFRP-3 induced insulin resistance.Taken together,we firstly demonstrated that intraperitoneally injected RFRP-3 can induce changes in appetite related factors in the hypothalamus of SD rats to promote the food intake accumulation,change meal microstructure and further lead to metabolic syndrome characterized by obesity and dyslipidaemia.Furthermore,the in vivo and morphological data we provided revealed the mechanism of RFRP-3 induced glucose metabolic disorders,suggesting that the occurrence and development of RFRP-3-induced diabetes may be related to hyperphagia,obesity,hyperglycemia,glucose intolerance,hypoinsulinism,hyperglucagon,insulin resistance and islet compensatory response.These results were not only provide basis for further investigation of physiological function of RFRP-3 on energy metabolism,but also provide a new way to explore the drug target of diabetes and obesity. |
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