Characterization of cholecystokinin producing enteroendocrine cells

CCK是一种肽类激素，主要由位于小肠近端的GI粘膜肠内分泌I细胞释放。CCK刺激胆囊收缩、胰腺外分泌、胃排空延迟和饱腹感。动物和人类的生理学研究表明，CCK分泌受饮食中长链脂肪酸、芳香族氨基酸和管腔CCK释放肽的刺激。 然而，CCK细胞感知管腔营养物质的精确分子基础仍不清楚。这主要是由于肠内分泌细胞（如CCK I细胞）相对稀少（<1%的粘膜细胞）且沿着GI道高度分散分布。缺乏一个忠实的细胞培养模型和缺乏一种方法来积累纯肠内分泌细胞一般和I细胞，特别是阻碍了在确定营养化学感受在胃肠道的分子基础的进展。 我们利用在CCK编码序列上游紧邻插入增强型绿色荧光蛋白（EGFP）基因的BAC转基因小鼠来鉴定产生CCK的I细胞。 用兔抗CCK抗体染色的胃肠道冰冻切片的免疫组织化学来评估转基因仅在CCK产生的I细胞中的忠实表达。 使用ImM EDTA和胶原酶分散小肠粘膜细胞以产生单细胞群。 通过荧光激活细胞分选（FACS）分离代表CCK产生I细胞的分散的EGFP阳性细胞。 从CCK-EGFP阳性I细胞的纯群体中提取的总RNA被逆转录并通过Affyssin小鼠基因组430-2.0基因芯片进行分析。 该相同的CCK-EGFP阳性I细胞制备物用于评估各种促分泌素对随后通过放射免疫测定法测量的CCK的刺激释放。 我们的研究表明，在CCK-EGFP BAC转基因小鼠中，EGFP在位于近端小肠上皮的CCK表达I细胞中忠实地表达。 通过表达EGFP的细胞的FACS，从分散的肠细胞中成功分离纯的（95%）表达CCK的I细胞。CCK表达细胞的基因表达谱的芯片研究表明，多个候选基因，如GPR 40，GPR 120，和钙敏感受体（CaR），可能作为营养刺激释放CCK的管腔传感器。 通过qRT-PCR验证CCK表达细胞中GPR 40和GPR 120转录物表达的存在。与我们的微阵列和qRT-PCR数据一致，FACS分离的纯CCK表达细胞响应于游离脂肪酸和苯丙氨酸以时间依赖性方式释放CCK。在用油酸管饲的WT和GPR 40 KO小鼠中的体内研究支持GPR 40作为LCFA的传感器的作用。

CCK is a peptide hormone released predominantly from GI mucosal enteroendocrine I cells located in the proximal small intestine. CCK stimulates gallbladder contraction, exocrine pancreatic secretion, delayed gastric emptying and satiety. Physiologic studies in animals and humans show that CCK secretion is stimulated by dietary medium to long chain fatty acids, aromatic amino acids and a luminal CCK releasing peptide. However, the precise molecular basis for CCK cell sensing of luminal nutrients remains unclear. This is largely due to the relative rarity (<1% of mucosal cells) and highly dispersed distribution of enteroendocrine cells, such as CCK I cells, along the GI tract. The absence of a faithful cell culture model and the lack of a method for accumulating pure enteroendocrine cells in general and I cells in particular has hindered progress in determining the molecular basis of nutrient chemosensation in the GI tract. We utilized a BAC transgenic mouse with the enhanced green fluorescent protein (EGFP) gene inserted immediately upstream of the CCK coding sequence to identify CCK producing I cells. Immunohistochemistry of gastrointestinal frozen sections stained with rabbit anti-CCK antibody was used to assess the faithful expression of the transgene only in CCK producing I cells. Small mucosal intestine cells were dispersed using 1mM EDTA and collagenase to produce a population of single cells. Dispersed EGFP positive cells representing CCK producing I cells was isolated by fluorescent activated cell sorting (FACS). Total RNA extracted from the pure population of CCK-EGFP positive I cells was reversed transcribed and analyzed by Affymetrix mouse genome 430-2.0 GeneChip. This same CCK-EGFP positive I cell preparation was utilized to assess a variety of secretagogues for stimulated release of CCK subsequently measured by radioimmunoassay. Our studies show that EGFP is faithfully expressed in CCK expressing I cells located in the proximal small intestinal epithelium in CCK-EGFP BAC transgenic mouse. Pure (95%) CCK expressing I cells were successfully isolated from dispersed intestinal cells via FACS of EGFP expressing cells. The gene expression profile of CCK expressing cells analyzed by microarray studies suggested multiple candidate genes, such as GPR40, GPR120, and the calcium sensing receptor (CaR) that may act as luminal sensors for nutrient stimulated release of CCK. The presence of GPR40 and GPR120 transcript expression in CCK expressing cells was verified by qRT-PCR. Consistent with our microarray and qRT-PCR data, FACS isolated pure CCK expressing cells release CCK in response to the free fatty acid and phenylalanine in a time dependent manner. In vivo studies in WT and GPR40 KO mice gavaged with oleic acid support the role of GPR40 as a sensor for LCFA.