Characterization of Gastrointestinal Ghrelin Producing Cells

由肠内分泌细胞分泌的多肽激素以孤立细胞的形式沿着胃肠道的粘膜扩散分布，调节食物的启动、消化、肠道运动和饱腹感。与从胰腺、脂肪组织和中枢神经系统释放的其他激素一起，这些肠道激素有助于保持体重、葡萄糖和能量的动态平衡。 在众多的胃肠道激素中，Ghrelin是一种由28个氨基酸组成的具有Ser3正辛酰化的多肽，由整个胃肠道的粘膜肠内分泌细胞分泌，其细胞密度在胃粘膜中最高，在肠道的尾部逐渐下降。与胃促生长素细胞密度的分布一致，65%至80%的循环中的胃促生长素是从胃中释放出来的。胃肠道内存在两种类型的Ghrelin细胞，一种是胃含氧酶黏膜中的闭合型细胞，没有管腔暴露；另一种是开放型细胞，其馀部分有顶端管腔接触。这两种类型的Ghrelin细胞提示，Ghrelin的分泌可能受到不同的调节，Ghrelin在胃肠道的不同区域可能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，胃促生长素水平在进食后迅速下降之前会上升。在这种阶段性释放的基础上，Ghrelin被推测在食物起始中发挥短期作用。在体重的长期调节中，循环中的Ghrelin水平与体重变化呈负相关。长期服用Ghrelin会增加体重，多种原因导致的体重减轻与Ghrelin水平升高有关。与瘦素和胰岛素类似，Ghrelin被认为是肥胖的信号。 目前，Ghrelin分泌的调节正在被深入研究，因为它在食欲障碍和体重异常方面具有治疗潜力。与葡萄糖和脂肪动态平衡有关的激素，如瘦素和胰岛素，已被研究为Ghrelin调节的候选激素。虽然已经有许多关于Ghrelin分泌调节的研究，但结果是多种多样的，没有一项涉及到在分子水平上直接调节Ghrelin产生细胞的分泌。 为了在分子水平上了解Ghrelin的直接调控，我们建立了转基因小鼠，在内源性Ghrelin启动子的控制下表达增强型绿色荧光蛋白(EGFP)，从而分离出纯净的Ghrelin产生细胞群。这些小鼠是健康的，可生育的，与它们的WT后代相比没有表现出任何表型。该转基因小鼠的抗Ghrelin免疫荧光研究证实，EGFP只在整个胃肠道的Ghrelin产生细胞中忠实地表达。用胶原酶/EDTA消化胃肠黏膜获得分散的单个细胞，进行荧光辅助细胞分选(FACS)，以产生纯的Ghrelin产生细胞群。将多种潜在的Ghrelin促分泌剂和抑制剂应用于急性培养的纯Ghrelin细胞，并用酰化Ghrelin免疫测定法测定它们对Ghrelin释放的影响。这些研究为胰高血糖素的直接刺激和瘦素抑制Ghrelin的分泌提供了证据。拥有一个纯的Ghrelin阳性细胞群体应该有助于我们询问参与Ghrelin分泌调节的潜在候选基因表达谱的能力。此外，Ghrelin产生细胞对EGFP的忠实表达表明，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。

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.