Simultaneous silencing of Npy and Dbh expression in hindbrain A1/C1 catecholamine cells suppresses glucoprivic feeding.
Simultaneous silencing of Npy and Dbh expression in hindbrain A1/C1 catecholamine cells suppresses glucoprivic feeding.
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DOI：
10.1523/jneurosci.4267-08.2009
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发表时间：
2009-01-07
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影响因子：
--
通讯作者：
Ritter S
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文献类型：
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作者：
Li AJ;Wang Q;Dinh TT;Ritter S
以前的数据强烈暗示后脑儿茶酚胺/神经肽Y（NPY）共表达神经元作为关键介质的葡萄糖消耗喂养反应。儿茶酚胺/神经肽Y细胞体集中在延髓腹外侧的A1和尾侧C1细胞群（A1/C1）中，这是一个对葡萄糖缺乏刺激高度敏感的区域。为了进一步研究这种儿茶酚胺亚群在葡萄糖调节中的重要性，我们使用小干扰RNA（siRNA）技术来产生NPY和多巴胺-β-羟化酶（DBH）（一种儿茶酚胺生物合成酶）的靶向基因敲除。与对照非靶向siRNA（ntRNA）侧相比，NPY siRNA和DBH siRNA（各0.02 nmol）的单侧注射均显著抑制靶基因的表达达2 d，如实时PCR所示，并降低蛋白表达达8 d，如免疫组化所示。随后，将靶向siRNA或对照NTRNA双侧注射到A1/C1中，并在3-7天后测试对2-脱氧-d-葡萄糖（2DG; 200 mg/kg）诱导的葡萄糖缺乏的反应。沉默无论是Npy或Dbh单独没有减少glucoprivic喂养或高血糖反应，与响应的ntRNA注射对照。相比之下，同时沉默Npy和Dbh使2DG诱导的摄食减少61%。无论是对2DG的高血糖反应，也不是由巯基乙酸（68 mg/kg）诱导的脂肪酸氧化阻断引起的进食（“lipoprivic喂养”），都不会因为这两个基因的同时沉默而减少。这些结果表明，儿茶酚胺和神经肽Y共同作用，以控制glucoprivic喂养和关键的神经肽Y/儿茶酚胺共表达神经元集中在A1/C1细胞群。
Previous data have strongly implicated hindbrain catecholamine/neuropeptide Y (NPY) coexpressing neurons as key mediators of the glucoprivic feeding response. Catecholamine/NPY cell bodies are concentrated in the A1 and caudal C1 cell cluster (A1/C1) in the ventrolateral medulla, a region highly sensitive to glucoprivic challenge. To further investigate the importance of this catecholamine subpopulation in glucoregulation, we used small interfering RNA (siRNA) technology to produce a targeted gene knockdown of NPY and dopamine-β-hydroxylase (DBH), a catecholamine biosynthetic enzyme. Unilateral injection of NPY siRNA and DBH siRNA (0.02 nmol each) both significantly inhibited expression of the targeted genes up to 2 d, as revealed by real-time PCR, and reduced protein expression up to 8 d, as revealed by immunohistochemistry, compared with the control nontargeting siRNA (ntRNA) side. Subsequently, targeted siRNA or control ntRNA was injected bilaterally into A1/C1 and responses to 2-deoxy-d-glucose (2DG; 200 mg/kg)-induced glucoprivation were tested 3–7 d later. Silencing of either Npy or Dbh alone did not reduce glucoprivic feeding or hyperglycemic responses, compared with responses of ntRNA-injected controls. In contrast, simultaneous silencing of both Npy and Dbh reduced 2DG-induced feeding by 61%. Neither the hyperglycemic response to 2DG nor feeding elicited by mercaptoacetate (68 mg/kg)-induced blockade of fatty acid oxidation (“lipoprivic feeding”) was reduced by simultaneous silencing of these two genes. These results suggest that catecholamines and NPY act conjointly to control glucoprivic feeding and that the crucial NPY/catecholamine coexpressing neurons are concentrated in the A1/C1 cell group.