Carnivorous fish have poor tolerance to carbohydrate in feed and low utilization rate of carbohydrate. Therefore, the balance of carbohydrate and lipids in the nutrient metabolism of carnivorous fish, the effective conversion and utilization of carbohydrate and lipids, and the feedback regulation of feeding are the key links for the efficient utilization of carnivorous fish feed. Carbohydrate response element binding protein (ChREBP) is a new transcription factor found in recent years in the glucose signaling pathway, and can also participate in feeding regulation. We performed in-vivo and in-vitro experiments to reveal the role of ChREBP in the glucose metabolism and feeding in mandarin fish. The mRNA expression of ChREBP and appetite regulatory factors were measured after intraperitoneal injection of glucose in mandarin fish Siniperca chuatsi and cotransfection with glucose and glucose+siRNA in the hypothalamic cells in mandarin fish. The results reveal that at hour 2 and 4 post intraperitoneal injection with 1 mg/g BW glucose, the blood glucose level of the mandarin fish increased significantly, but food intake decreased significantly, and it also displayed a significantly increased ChREBP mRNA expression levels in liver. At hour 4 post injection, hypothalamic ChREBP mRNA level was significantly increased, whereas the mRNA expression levels of appetite genes neuropeptide Y (npy) and agouti-related peptide (AgRP) were decreased significantly. When the glucose concentration was 40 mmol/L, the expression level of ChREBP mRNA in mandarin fish hypothalamic cells was significantly up-regulated, but the expression level of appetite gene npy mRNA was down-regulated. When siRNA and glucose were co-transfected into mandarin fish brain cells, the expression level of chrebp mRNA was significantly decreased, and the appetite gene npy mRNA was significantly increased. The results indicated that glucose regulated food intake through the modulation of appetite gene npy by ChREBP.

为了建立可行、高效的下丘脑神经元细胞技术,本研究通过分离鳜下丘脑组织,用Ⅰ型胶原酶将组织消化成单细胞悬液,并用完全培养液进行培养,在培养的第3、4、5和6天观察细胞的形态.结果 显示,培养第3天时细胞贴壁量少,胞体小并且呈单个分布;在培养第4天,细胞的数量显著增多,且具有典型的神经元的形态,胞体饱满;第5天神经元胞体的融合度进一步增大,突起增长增粗,许多分支互相交错连接而形成密集的神经纤维网络;第6天细胞活性减弱,开始凋亡.使用CCK-8法检测细胞活性,结果显示,在培养第5天,细胞活性最高;采用荧光定量PCR(RT-PCR)法和免疫荧光鉴定法对神经元细胞进行鉴定,经RT-PCR检测发现,培养的下丘脑细胞可以表达神经元特异基因noggin,经免疫荧光鉴定,下丘脑神经元细胞纯度为95.9％.研究表明,通过此培养方法能够获得纯度较高的鳜下丘脑神经元细胞,为进一步在细胞水平研究鳜的摄食与能量代谢相关机理奠定基础.

Taste receptors play important roles in perception of L-amino acids and feeding behavior. However, convincing evidence providing causal links between taste receptors and feeding habits is lacking. We generated two mutant lines with t1r1 deletion in zebrafish by CRISPR/Cas9 technology. t1r1 deletion eliminated sensitivity to alanine in zebrafish. Additionally, it did not affect feeding behavior of zebrafish supplied with habitual food such as Artemia salina, while significantly enhanced their acceptance of rejective food such as duckweed and plantprotein source diets. Furthermore, replacement of fish meal with plant protein in diets significantly suppressed transcript levels of orexigenic gene npy and increased mRNA expression levels of anorexigenic gene pomca in wild-type zebrafish but had no effect in t1r1- /- zebrafish. In addition, when fed with fish meal diet, transcript levels of npy and pomca were significantly different in t1r1-/- zebrafish compared with wild-type zebrafish. Overall, these findings suggested that T1R1 was indispensable for amino acid perception and significantly affected the feeding preference and habit in zebrafish.

以鳜(Siniperca chuatsi)为研究对象,使用cDNA 3′末端快速克隆法扩增瘦素受体基因(lepr)cDNA序列,获得了由mRNA 3′端可变剪切产生的鳜lepr的4个不同亚型,包括编码序列(coding sequence,CDS)长度为3474 bp、编码1157个氨基酸的长型受体亚型lepr-L,以及3个短型受体亚型lepr-S1、lepr-S2和lepr-S3,CDS长度分别为1512、945和915 bp,分别编码503、314、304个氨基酸.对氨基酸序列进行结构域分析和多重比对发现,鳜lepr长型受体亚型包含完整的功能域,短型受体亚型无跨膜区及胞内结构,鳜lepr及其leptin结合域(leptin binding domain,LBD)序列保守程度高.鳜lepr在鳃中表达最高,其次是肾和垂体,腹腔注射鳜leptin B而非leptin A的同源重组蛋白2 h后引起脑lepr表达量的升高(P<0.05).以上结果表明,鳜leptins能引起组织中lepr表达的不同变化而发挥独特的生理功能.

为了探讨6-磷酸葡萄糖酶催化亚基(glucose-6-phosphatase catalytic subunit,G6PC)在草鱼(Ctenopharyngodon idellus)糖代谢中的作用,采用同源序列比对的方式,在草鱼基因组中获取了3个g6pc基因的序列,通过序列比对和进化树分析,将其分别命名为g6pca、g6pcb1和g6pcb2,其编码的氨基酸序列与斑马鱼(Danio rerio)、虹鳟(Oncorhynchus mykiss)和人(Homo sapiens)具有较高的同源性,相似度分别为85％～94％、64％～83％和54％～66％.同线性分析表明g6pc在草鱼染色体上的分布与斑马鱼等高度相似,表明草鱼g6pc基因在进化中具有较高的保守性.利用RT-PCR检测3个基因在鳃、脂肪、脑、心脏、肝、肾、前肠、中肠、后肠和肌肉10个组织中的表达,结果显示,g6pca在脑和肝中表达量较高,脂肪组织次之;g6pcb1在肝中表达量最高,中肠次之;g6pcb2在心脏表达量最高,其次为脂肪组织.同时探讨了高糖饲料对草鱼不同g6pc亚型转录水平的影响,以及不同浓度葡萄糖和胰岛素刺激草鱼肝细胞(L8824)后对g6pca转录水平的影响.结果显示,饲喂高糖饲料(7周)后,与对照组相比,草鱼肝脏g6pca mRNA水平显著升高,g6pcb1和g6pcb2 mRNA水平无显著变化.离体情况,与5 mmol/L葡萄糖组相比,15 mmol/L葡萄糖显著增加了L8824的g6pca mRNA水平,且1 mol/L胰岛素可以抑制这种作用;30 mmol/L葡萄糖对L8824 g6pca mRNA水平无显著性影响.本研究表明,草鱼g6pc发生加倍后存在功能分化,高糖可以诱导g6pca mRNA的表达,而对g6pcb1和g6pcb2的转录水平无影响,其具体功能还需进一步研究.