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: 8349819
• 负责人: Stephen Wank
• 金额: $ 7.58万
• 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/8349819
• 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; Edetic Acid; 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.

肠内分泌细胞分泌的肽激素作为独立细胞沿着胃肠道粘膜广泛分布，协调调节食物起始、消化、肠道蠕动和饱腹感。这些肠道激素与胰腺、脂肪组织和中枢神经系统释放的其他激素一起有助于维持体重、葡萄糖和能量稳态。 在众多胃肠道肽激素中，生长素释放肽（ghrelin）是一种具有 Ser3 正辛酰化的 28 个氨基酸肽，由整个胃肠道的粘膜肠内分泌细胞分泌，细胞密度在胃粘膜中最高，沿肠道尾部逐渐下降。 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，一种是胃泌酸粘膜中的封闭型细胞，不暴露于腔内，另一种是肠道其余部分中与顶端腔接触的开放型细胞。 这两种类型的生长素释放肽细胞表明生长素释放肽的分泌可能受到不同的调节，并且生长素释放肽可能在胃肠道的不同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，饥饿素水平在进餐前会上升，摄入后会迅速下降。 基于这种阶段性释放，生长素释放肽被推测在食物启动中发挥短期作用。在体重的长期调节中，循环中的生长素释放肽水平与体重变化成反比。长期服用生长素释放肽会增加体重，多种原因导致的体重减轻与生长素释放肽水平升高有关。 与瘦素和胰岛素类似，生长素释放肽被认为是肥胖信号。 目前，人们正在深入研究生长素释放肽分泌的调节，以了解其在食欲障碍和体重异常方面的治疗潜力。 参与葡萄糖和脂肪稳态的激素，例如瘦素和胰岛素，已作为生长素释放肽调节的候选激素进行了研究。虽然已经有许多关于生长素释放肽分泌调节的研究，但结果各不相同，并且没有一个研究在分子水平上解决生长素释放肽产生细胞分泌的直接调节。 为了在分子水平上了解生长素释放肽的直接调节，我们培育了转基因小鼠，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），从而可以分离出纯的生长素释放肽产生细胞群。这些小鼠健康、有生育能力，与 WT 同窝小鼠相比没有表现出任何表型。 在该转基因小鼠系中进行的抗生长素释放肽免疫荧光研究证实，EGFP 仅在整个胃肠道中产生生长素释放肽的细胞中忠实表达。通过胶原酶/EDTA消化胃肠粘膜获得的分散的单细胞进行荧光辅助细胞分选（FACS）以产生纯的生长素释放肽产生细胞群。 将多种潜在的生长素释放肽促分泌剂和抑制剂应用于急性培养的纯生长素释放肽细胞，并通过酰化生长素释放肽的免疫测定来测量它们对生长素释放肽释放的影响。 这些研究为胰高血糖素直接刺激生长素释放肽和瘦素抑制生长素释放肽分泌提供了证据。拥有纯的生长素释放肽阳性细胞群应该有助于我们询问基因表达谱以寻找参与生长素释放肽分泌调节的潜在候选者的能力。 此外，Ghrelin 产生细胞忠实表达 EGFP 表明该启动子区域可进一步用于生成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。