Characterization of Gastrointestinal Ghrelin Producing Cells

胃肠道生长素释放肽产生细胞的表征

• 批准号: 7734189
• 负责人: Stephen Wank
• 金额: $ 22.94万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734189
• 关键词: Address; Adipose tissue; Affect; Amino Acids; Apical; Appetite Disorder; Body Weight; Body Weight Changes; Body Weight decreased; Cell Density; Cell Separation; Cells; Chronic; Development; Digestion; Enteroendocrine Cell; Fluorescence; Food; Future; Gastric mucosa; Gastrointestinal tract structure; Gene Expression Profile; Genes; Glucagon; Glucose; Homeostasis; Hormones; Human; Immunoassay; Immunofluorescence Immunologic; In Vitro; Ingestion; Insulin; Intestines; Leptin; Mammals; Measures; Methods; Molecular; Morphology; Mucous Membrane; Mus; Neuraxis; Obesity; Pancreas; Peptides; Phenotype; Physiological; Play; Population; Preparation; Promoter Regions; Regulation; Role; Satiation; Signal Transduction; Stomach; Therapeutic; Tissues; Transgenic Mice; Transgenic Organisms; Weight Gain; base; cell motility; collagenase; enhanced green fluorescent protein; falls; gastrointestinal; ghrelin; inhibitor/antagonist; loss of function; mouse model; peptide hormone; promoter

Peptide hormones secreted by enteroendocrine cells diffusely distributed as isolated cells along the mucosa of the GI tract orchestrate to regulate food initiation, digestion, gut motility and satiation. Along with other hormones released from pancreas, adipose tissue and the central nervous system, these gut hormones help to maintain body weight, glucose and energy homeostasis. Among the many GI peptide hormones, ghrelin, a 28-amino acid peptide with a Ser3 n-octanoylation, is secreted from mucosal enteroendocrine cells throughout the GI tract with a cell density that is highest in the gastric mucosa and declines caudally along the intestine. Consistent with the distribution of ghrelin cell density, 65 to 80% of circulating ghrelin is released from the stomach. Two types of ghrelin cells exist in the GI tract, a closed-type in the oxyntic mucosa of the stomach with no luminal exposure and an open-type with apical luminal contact in the remainder of the gut. These two types of ghrelin cells suggest that ghrelin secretion may be differentially regulated and that ghrelin may play different physiological roles in various regions of the GI tract. The physiological functions of ghrelin have been extensively studied. In both humans and other mammals, ghrelin levels rise before a meal fall rapidly after ingestion. On the basis of this phasic release, ghrelin has been speculated to play a short term role in food initiation. In long-term regulation of body weight, circulating ghrelin levels correlate inversely with body weight change. Chronic administration of ghrelin increases body weight and weight loss resulting from multiple causes is associated with elevated ghrelin levels. Similar to leptin and insulin, ghrelin has been postulated to be an adiposity signal. Currently, regulation of ghrelin secretion is being intensely investigated for its therapeutic potential in disorders of appetite and abnormalities of body weight. Hormones involved in glucose and adipose homeostasis, such as leptin and insulin, have been investigated as candidates for ghrelin regulation. While there have been many studies on the regulation of ghrelin secretion, the results have been variable and none have addressed the direct regulation of secretion from ghrelin producing cells at the molecular level. To understand the direct regulation ghrelin at the molecular level, we generated transgenic mice in which expression of enhanced green fluorescent protein (EGFP) under the control of the endogenous ghrelin promoter, allows the isolation of a pure population of ghrelin producing cells. These mice were healthy, fertile and displayed no phenotype compared to their WT littermates. Anti-ghrelin immunofluorescence studies in this transgenic mouse line confirmed that EGFP is faithfully expressed only in the ghrelin producing cells throughout GI tract. Dispersed single cells obtained by collagenase/EDTA digestion of gastrointestinal mucosa were subjected to fluorescence-assisted cell sorting (FACS) to produce a pure population of ghrelin producing cells. A variety of potential ghrelin secretagogues and inhibitors were applied to the acutely cultured pure ghrelin cells and their affect on ghrelin release measured by immunoassay for acylated ghrelin. These studies provide evidence for the direct stimulation by glucagon and inhibition by leptin of ghrelin secretion. Having a pure population of ghrelin-positive cells should facilitate our ability to interrogate the gene expression profile for potential candidates involved in the regulation of ghrelin secretion. In addition, the faithful expression of EGFP by ghrelin producing cells indicates that this promoter region can be further employed to generate both tissue-specific gain or loss-of-function transgenic mouse models for future studies.

由肠内分泌细胞分泌的肽激素作为分离的细胞沿胃肠道粘膜沿着弥散分布，协调调节食物起始、消化、肠道运动和饱足。沿着从胰腺、脂肪组织和中枢神经系统释放的其他激素，这些肠道激素有助于维持体重、葡萄糖和能量稳态。 在许多GI肽激素中，生长素释放肽（ghrelin），一种具有Ser 3正辛酰化的28-氨基酸肽，从整个GI道的粘膜肠内分泌细胞分泌，其细胞密度在胃粘膜中最高，并沿肠向尾侧沿着下降。 与生长素释放肽细胞密度的分布一致，65%至80%的循环生长素释放肽从胃中释放。胃肠道中存在两种类型的生长素释放肽细胞，一种是在胃的泌酸粘膜中的封闭型，没有腔暴露，另一种是在肠的其余部分中的开放型，具有顶端腔接触。 这两种类型的生长激素释放肽细胞表明，生长激素释放肽分泌可能受到差异调节，生长激素释放肽可能在胃肠道的各个区域发挥不同的生理作用。 Ghrelin的生理功能已被广泛研究。 在人类和其他哺乳动物中，胃饥饿素水平在饭前上升，在摄入后迅速下降。 基于这种阶段性释放，推测ghrelin在食物起始中起短期作用。在体重的长期调节中，循环饥饿素水平与体重变化呈负相关。长期施用生长素释放肽增加体重，并且由多种原因引起的体重减轻与生长素释放肽水平升高相关。 与瘦素和胰岛素类似，生长激素释放肽被认为是肥胖的信号。 目前，ghrelin分泌的调节正因其在食欲障碍和体重异常中的治疗潜力而被深入研究。 参与葡萄糖和脂肪稳态的激素，如瘦素和胰岛素，已被研究为生长激素释放肽调节的候选者。虽然已经有许多关于生长素释放肽分泌调节的研究，但是结果是可变的，并且没有一个研究在分子水平上直接调节生长素释放肽产生细胞的分泌。 为了在分子水平上理解生长素释放肽的直接调节，我们产生了转基因小鼠，其中在内源性生长素释放肽启动子的控制下表达增强型绿色荧光蛋白（EGFP），允许分离生长素释放肽产生细胞的纯群体。这些小鼠是健康的、可生育的，并且与其WT同窝小鼠相比未显示表型。 在该转基因小鼠系中的抗生长素释放肽免疫荧光研究证实，EGFP仅在整个胃肠道的生长素释放肽产生细胞中忠实地表达。对通过胶原酶/EDTA消化胃肠道粘膜获得的分散的单细胞进行荧光辅助细胞分选（FACS）以产生生长素释放肽产生细胞的纯群体。 将各种潜在的ghrelin促分泌剂和抑制剂应用于急性培养的纯ghrelin细胞，并通过免疫测定法测定其对ghrelin释放的影响。 这些研究为胰高血糖素直接刺激和瘦素抑制ghrelin分泌提供了证据。有一个纯粹的群体生长激素释放肽阳性细胞应有助于我们的能力，询问基因表达谱的潜在候选人参与调节生长激素释放肽分泌。 此外，通过生长素释放肽产生细胞忠实地表达EGFP表明，该启动子区域可以进一步用于产生组织特异性获得或功能丧失的转基因小鼠模型，用于未来的研究。