| Ghrelin is produced by endocrine cells in the oxyntic mucosa of the stomach. Structurally, ghrelin resembles motilin, which is a 28-amino-acid peptide as an endogenous ligand for the growth hormone (GH) secretagogue receptor with molecular weight 3310. Ghrelin has two isoforms including unacylated and acylated ghrelin. Except for the relation to the release of GH, effects on glucose metabolism, food intake, cell proliferation and suppressing inflammatory cytokines, it also exert effects on promoting gastric motility and accelerating gastric emptying. However, physiologically, plasma ghrelin is elevated following prolonged fasting and levels are suppressed after eating. Hence it is unlikely to play a role in the regulation of postprandial gastric emptying.Dietary fibers are complex carbohydrates consisting of both soluble and insoluble components. The insoluble fibers have important bulking properties, whereas the soluble forms can be fermented by certain species of gut bacteria, leading to physiologically active byproducts. Short chain fatty acids (SCFAs) are among the most abundant of these dietary metabolites, including acetate, propionate and butyrate, which have a crucial role as a fuel source for intestinal epithelial cells and exert effects on gut morphology and function. These effects were mostly played via GPR41 and GPR43.Petersen et al presented a novel in vitro platform to generate functional L cells from three-dimensional cultures of mouse and human intestinal crypts by adding SCFAs to promote much secretion of GLP-1.The intestinal epithelium constitutes a system of constant and rapid renewal triggered by proliferation of intestinal stem cells (ISCs), and is an ideal system for studying cell proliferation, migration, and differentiation. Recently, Sato and colleagues have established long-term culture conditions under which single crypts or isolated stem cells from the stomach, small intestine, or colon grow to form crypt/glandular structures that expand via continual fission events, while continuously producing all of the differentiated cell types specific to the tissue of origin. These three-dimensional epithelial structures were originally called "organoids," but to avoid confusion among tissues and to distinguish these cultures from previous "organoids" composed of crypts and pericryptal myofibroblasts, epithelial organoids from the stomach are called gastroids, those from the small intestine are called enteroids, and those from the colon are called colonoids. These experimental model systems constitute useful tools for studying the regulation of gastrointestinal stem cells as well as the proliferation and the differentiation of the intestinal epithelial cells throughout the digestive tract.Disorders associated with delayed gastric emptying are common. They include patients with diabetic or idiopathic gastroparesis, certain patients with functional dyspepsia and those recovering from abdominal surgery or receiving enteral feeding. Currently, ghrelin has been proved to exert effect on the regulation of phase â…¢-like contractions in stomach and accelerating gastric emptying. Therefore, this study presented a novel in vitro platform to generate functional ghrelin producing cells (GPC) from 3D cultures of mouse gastroids that were maintained in culture for 3 wks then incubated with SCFA mixture to investigate the effect of SCFAs on GPC and the mechanism. At the same time, we studied the effects of high fiber food and SCFAs on the expression of ghrelin in stomach and serum and gastric empting.The main results of our research are as follows:1. In vitro study1. To understand the effect of SCFAs on the GPCs, we presented a novel in vitro platform to generate functional GPCs from 3D cultures of mouse and human gastroids that were maintained in culture for 3 wks. To test the effect of SCFAs on GPC differentiation, a combination of acetate, propionate, and butyrate (5,1, and 1 mM, respectively) was added to the culture medium. For control mouse gastroids, regular medium with linoleic acid (150uM) and regular medium without SCFAs were used.24 hours later, we found that SCFAs resulted in a 6-8 fold increase in ghrelin gene expression compared to CT and LFA group both in mouse and human gastroids.2. SCFAs had been long known to play stimulating effects via GPR41 and GPR43.To test which receptor play the domain effect, we used GPR41 agonist and GPR43 agonist treat mouse and human gastroids and found that GPR43 agonist resulted in almost 6-12 fold increase in ghrelin gene expression compared to CT group and GPR41 agonist had no effect on ghrelin gene expression in mice and human gastroids. Immunostaining of ghrelin in frozen sections of mice gastroids showed that the number of GPCs increased after GPR43 agonist treatment but did not change after GPR41 agonist treatment. At the same time, almost 90% of GPCs expressed GPR43. Therefore, the stimulating effect of SCFAs on the differentiation of GPCs appeared to be mediated by GPR43 receptor in mouse and human gastroids.3. To test which SCFA paly the stimulating effect on GPCs differentiation, we use acetate (5mM), propionate (1mM) and butyrate (1mM) separately to stimulate the mouse and human gastroids. The RT-PCR results showed that butyrate significantly increased ghrelin gene expression (P=0.001) 8.46 times in mouse gastroids and 4.68 times (P=0.01) in human gastroids. Immunostaining of ghrelin in frozen sections of mice gastroids showed that the number of GPCs increased after butyrate treatment. Therefore, butyrate was the only potent promoter of the differentiation of GPCs in mouse and human gastroids.4. To test the effect of butyrate on differentiation of the gastric cell lineages, we performed marker gene expression analysis. In mouse and human organoids, buyrate treatment had no effect on expression of MUC5AC (gastric surface mucous cell), TFF2 (antral gland cell), ATP4A (parietal cell) and MIST1 (chief cell) but the neuroendocrine cell marker ChgA. In accordance with our data on ghrelin content, gastric progenitor cell marker SOX9 and neuroendocrine cell marker ChgA were elevated in mouse and human organoids compared with the control which is related to cell differentiation. Therefore, we thought that butyrate is the specific SCFA to promote ghrelin producing cells differentiation in mouse and human gastroids via GPR43.2. In vivo study 1. We have proved that butyrate is the specific SCFA to promote ghrelin producing cells differentiation in mouse and human gastroids via GPR43. To test the effect of SCFA on the differentiation of GPCs in vivo, we used 2 week high fiber and butyrate drinking model. Mice were fed with normal chow, high fiber diet (pectin,30%) or normal chow with butyrate drinking (100 mM) 2 weeks. RT-PCR and western blot results also showed the expression of ghrelin in mice stomach increased after high fiber diet and butyrate drinking in physiological state. Enzyme-Linked ImmunoSorbent Assay (ELISA) test also found that high fiber diet and butyrate drinking can increase the concentration of active ghrelin in serum which is responded for gastric motility.2. At the same time, we did gastric emptying study in three groups including mice fed with normal chow, high fiber and butyrate drinking.2 weeks later, mice were fasted 20 hours and then underwent gastric emptying experiment. The results showed that mice in high fiber diet and butyrate drinking group emptied more than control group. Therefore, high fiber diet and butyrate drinking can accelerate gastric emptying in mice.3. To test if the increase of ghrelin promoting gastric emptying in mice, mice with high fiber and butyrate drinking group were treated with ghrelin antagonist ([D-Lys-3]-GHRP-6,40nmol, intraperitoneally injection). Gastric emptying results showed butyrate can accelerate the gastric emptying in mice which can be blocked by ghrelin antagonist. At the same time, high fiber diet can accelerate the gastric emptying in mice which can be blocked by ghrelin antagonist.In conclusion, butyrate is the specific SCFA to promote ghrelin producing cells differentiation in mouse and human gastroids via GPR43. At the same time, high fiber diet and butyrate can increase the expression of ghrelin in mice stomach the concentration of active ghrelin in serum which is responded for gastric motility in physiological state. This study may supply new insight of therapy of gastric motility dysfunction. |
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