Ghrelin和SHU9119通过促进CART神经元调节小鼠采食
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摘要
为研究Ghrelin和SHU9119如何作用于小鼠下丘脑可卡因和苯丙胺转录调节因子(CART)神经元参与小鼠采食调控,本试验以免疫荧光技术为主,利用代谢笼和冰冻切片的方式获取试验材料,综合小鼠采食量测定,CART神经元表达数目的统计学分析,及CART神经元、刺鼠相关蛋白(AgRP)神经元同黑皮质素4型受体(MC4R)神经元间共定位信息,对Ghrelin和SHU9119通过不同方式介导CART神经元以参与小鼠采食调控这一作用机理进行初步阐述。结果显示:健康4周龄C57BL/6雄性小鼠,腹腔注射Ghrelin(50μg/kg,n=3)后,其下丘脑背内侧核(DMH)区域CART神经元表达数目显著上升(P<0.05);而SHU9119(50μg/kg,n=5)则显著促进弓状核(ARC)区域的CART神经元的表达(P<0.01),2种药物促进CART神经元表达的部位并不相同。同时,2种药物的单独注射均可对小鼠采食活动产生一定的促进作用(Ghrelin组n=7;SHU9119组n=7)。有趣的是,CART神经元在DMH区域同AgRP神经元之间具有突触联系,而CART神经元与表达MC4R的神经元之间的共表达关系则发生在ARC区域。结合已有的研究证据,我们推测Ghrelin和SHU9119可能通过不同方式参与对CART神经元表达的调节,并最终影响小鼠采食活动。期望通过本研究为今后进一步探明外周信号分子如何参与促厌食神经元对机体能量代谢调控提供新的思路和证据。
In this study,immunofluorescence technique,the curve of mice food intake and the expression of CART neurons were used for analysis.And the mechanism of Ghrelin and SHU9119 active CART neurons affects on CART neurons to regulate mice food intake has been described.The results shown that,in 4 week age male mice,after injecting ghrelin(50μg/kg,n=3),CART neurons expression was promoted in DMH nucleus(P<0.05)and CART neuron expression was enhanced in ARC nucleus when injected SHU919 alone(50μg/kg,n=5,P<0.01).But Ghrelin and SHU9119 had different acted nucleus area in mice hypothalamic.At the same time,there was both increased food intake of male mice when injected Ghrelin or SHU919 alone(ghrelin group n=7;SHU9119 group n=7).Surprisingly,CART neuron had co-localized with the axon of AgRP neuron in DMH nucleus,while also co-expressed with MC4 R-positive neuron in ARC nucleus.Combined with previous research,all of our results showed that Ghrelin and SHU9119 have variety ways promoting CART neuron expression,and affect mice food intake,finally.These studies provide evidence in the study of energy metabolism regulation link peripheral signaling molecules to anorectic neurons.
In this study,immunofluorescence technique,the curve of mice food intake and the expression of CART neurons were used for analysis.And the mechanism of Ghrelin and SHU9119 active CART neurons affects on CART neurons to regulate mice food intake has been described.The results shown that,in 4 week age male mice,after injecting ghrelin(50μg/kg,n=3),CART neurons expression was promoted in DMH nucleus(P<0.05)and CART neuron expression was enhanced in ARC nucleus when injected SHU919 alone(50μg/kg,n=5,P<0.01).But Ghrelin and SHU9119 had different acted nucleus area in mice hypothalamic.At the same time,there was both increased food intake of male mice when injected Ghrelin or SHU919 alone(ghrelin group n=7;SHU9119 group n=7).Surprisingly,CART neuron had co-localized with the axon of AgRP neuron in DMH nucleus,while also co-expressed with MC4 R-positive neuron in ARC nucleus.Combined with previous research,all of our results showed that Ghrelin and SHU9119 have variety ways promoting CART neuron expression,and affect mice food intake,finally.These studies provide evidence in the study of energy metabolism regulation link peripheral signaling molecules to anorectic neurons.
引文
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[8]MORTENSEN A H,CAMPER S A.Cocaine-and amphetamine regulated transcript(cart)peptide is expressed in precursor cells and somatotropes of the mouse pituitary gland[J].PLoS ONE,2016,11(9):e160068.
[9]MULLER T D,NOGUEIRAS R,ANDERMANN M L,et al.Ghrelin[J].Mol Metabol,2015,4(6):437-460.
[10]BALTHASAR N,DALGAARD L T,LEE C E,et al.Divergence of melanocortin pathways in the control of food intake and energy expenditure[J].Cell,2005,123(3):493-505.
[11]STARK R,ASHLEY S E,ANDREWS Z B.AMPK and the neuroendocrine regulation of appetite and energy expenditure[J].Mol Cell Endocrinol,2013,366(2):215-223.
[12]SHAH B P,VONG L,OLSON D P,et al.MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus[J].Proceed Nat Acad Sci,2014,111(36):13193-13198.
[13]SUTTON G M,JOSEPHINE B M,GU X,et al.A derivative of the melanocortin receptor antagonist SHU9119(PG932)increases food intake when administered peripherally[J].Peptides,2008,29(1):104-111.
[14]GUL TIRYAKI-SONMEZ S V B O.Obesity,food intake and exercise:relationship with ghrelin[J].Biom Human Kinetics,2015(7):119-127.
[15]RODRIGUEZ E M,BLAZQUEZ J L,GUERRA M.The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus:the former opens to the portal blood and the latter to the cerebrospinal fluid[J].Peptides,2010,31(4):757-776.
[16]FRY M,FERGUSON A V.Ghrelin:central nervous system sites of action in regulation of energy balance[J].Internat J Peptides,2010,2010:1-8.
[17]WANG Q,LIU C,UCHIDA A,et al.Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin[J].Mol Metabol,2014,3(1):64-72.
[18]WAN Y,ZHANG Y,JI P,et al.Molecular characterization of CART,AgRP,and MC4R genes and their expression with fasting and refeeding in common carp(Cyprinus carpio)[J].Mol Biol Rep,2012,39(3):2215-2223.
[2]DOUGLASS J,DAOUD S.Characterization of the human cDNA and genomic DNA encoding CART:a cocaine and amphetamine-regulated transcript[J].Gene,1996,169(2):241-245.
[3]LAU J,HERZOG H.CART in the regulation of appetite and energy homeostasis[J].Front Neurosci,2014(12):8.
[4]LAMBERT P D,COUCEYRO P R,MCGIRR K M,et al.CART peptides in the central control of feeding and interactions with neuropeptide Y[J].Synapse,1998,29(4):293-298.
[5]WIERUP N,RICHARDS W G,BANNON A W,et al.CART knock out mice have impaired insulin secretion and glucose intolerance,altered beta cell morphology and increased body weight[J].Regul Pept,2005,129(1/3):203-211.
[6]MOFFETT M,STANEK L,HARLEY J,et al.Studies of cocaine and amphetamine-regulated transcript(CART)knockout mice[J].Peptides,2006,27(8):2037-2045.
[7]MENYHERT J,WITTMANN G,LECHAN R M,et al.Cocaine and amphetamine-regulated transcript(CART)is colocalized with the orexigenic neuropeptide Y and agouti-related protein and absent from the anorexigenic alpha-melanocyte-stimulating hormone neurons in the infundibular nucleus of the human hypothalamus[J].Endocrinology,2007,148(9):4276-4281.
[8]MORTENSEN A H,CAMPER S A.Cocaine-and amphetamine regulated transcript(cart)peptide is expressed in precursor cells and somatotropes of the mouse pituitary gland[J].PLoS ONE,2016,11(9):e160068.
[9]MULLER T D,NOGUEIRAS R,ANDERMANN M L,et al.Ghrelin[J].Mol Metabol,2015,4(6):437-460.
[10]BALTHASAR N,DALGAARD L T,LEE C E,et al.Divergence of melanocortin pathways in the control of food intake and energy expenditure[J].Cell,2005,123(3):493-505.
[11]STARK R,ASHLEY S E,ANDREWS Z B.AMPK and the neuroendocrine regulation of appetite and energy expenditure[J].Mol Cell Endocrinol,2013,366(2):215-223.
[12]SHAH B P,VONG L,OLSON D P,et al.MC4R-expressing glutamatergic neurons in the paraventricular hypothalamus regulate feeding and are synaptically connected to the parabrachial nucleus[J].Proceed Nat Acad Sci,2014,111(36):13193-13198.
[13]SUTTON G M,JOSEPHINE B M,GU X,et al.A derivative of the melanocortin receptor antagonist SHU9119(PG932)increases food intake when administered peripherally[J].Peptides,2008,29(1):104-111.
[14]GUL TIRYAKI-SONMEZ S V B O.Obesity,food intake and exercise:relationship with ghrelin[J].Biom Human Kinetics,2015(7):119-127.
[15]RODRIGUEZ E M,BLAZQUEZ J L,GUERRA M.The design of barriers in the hypothalamus allows the median eminence and the arcuate nucleus to enjoy private milieus:the former opens to the portal blood and the latter to the cerebrospinal fluid[J].Peptides,2010,31(4):757-776.
[16]FRY M,FERGUSON A V.Ghrelin:central nervous system sites of action in regulation of energy balance[J].Internat J Peptides,2010,2010:1-8.
[17]WANG Q,LIU C,UCHIDA A,et al.Arcuate AgRP neurons mediate orexigenic and glucoregulatory actions of ghrelin[J].Mol Metabol,2014,3(1):64-72.
[18]WAN Y,ZHANG Y,JI P,et al.Molecular characterization of CART,AgRP,and MC4R genes and their expression with fasting and refeeding in common carp(Cyprinus carpio)[J].Mol Biol Rep,2012,39(3):2215-2223.