The nutrient-sensing mechanisms of the mouse stomach and the ghrelin cell in health and obesity

Abstract

Background: Ghrelin is a gastric hormone with multiple physiological functions, including the stimulation of food intake and adiposity. It is well established that circulating ghrelin levels drop after food intake, however, the mechanisms involved in the reduction of postprandial circulating ghrelin levels are not fully understood. In this regard, the ability of the gastrointestinal tract to detect nutrients is critical in the modulation of gut hormone secretion, with the detection of nutrients performed by specialised nutrient chemosensors located on enteroendocrine cells. However, limited information is available on the nutrient-sensing capabilities of the stomach. This PhD project explored the expression of nutrient chemosensors of the mouse stomach and gastric ghrelin cells in health and obesity. Further, the role of nutrients and nutrient chemosensors in the secretion of gastric ghrelin was also investigated. Aims: 1) Investigate the expression of nutrient chemosensors in different regions of the mouse stomach, with particular emphasis on co-expression with ghrelin. 2) Assess the role of nutrients and nutrient chemosensors in the secretion of gastric ghrelin. 3) Determine the effect of high-fat diet (HFD)-induced obesity on the expression of nutrient chemosensors in the mouse stomach and the level of co-expression with ghrelin. Methods and results: The study presented in Chapter 2 characterised the mRNA expression of a repertoire of nutrient chemosensors (protein: GPR93, CaSR, MGluR4; fatty acids: CD36, FFAR2&4; sweet/umami: T1R3, tastetransduction components (TRPM5, GNAT2&3) in the gastric antrum and corpus of the mouse stomach. In addition, immunofluorescence experiments determined the protein expression of GPR93, T1R3, FFAR4 and CD36 in both gastric regions, and their degree of co-expression with ghrelin. Results from this chapter showed that the majority of nutrient chemosensors presented higher mRNA levels in the antrum than corpus, with a similar regionality observed at the protein level. Moreover, co-expression studies showed that at least 60% of ghrelin-positive cells expressed T1R3 and FFAR4, and over 80% expressed GPR93 and CD36. Chapter 3 extended this investigation by 1) assessing the secretion of total ghrelin (TG) and acyl ghrelin (AG) in response to a wide range of nutrients (2 and 20 mM D-glucose, 20 mM L-phenylalanine, 5% protein hydrolysate (peptone), 5% D-mannitol, 2 mM α-linolenic acid and 5% fat emulsion (Intralipid)), and 2) determining the role of FFAR4 and CD36 in the α -linolenic acid and 5% intralipid-dependent secretion of TG and AG. Results from Chapter 3 showed that TG and AG secretion from the mouse stomach was modulated in a nutrient-specific manner. Glucose and mannitol did not affect TG and AG secretion. Peptone stimulated TG and AG secretion, while intralipid simultaneously reduced TG and stimulated AG secretion. L-phenylalanine and α-linolenic acid reduced AG release, without changing TG release. Moreover, the modulation of TG and AG secretion by α -linolenic acid and intralipid was independent of FFAR4 and CD36 activation. Chapter 4 determined the mRNA expression of gastric nutrient chemosensors (i.e. same repertoire investigated in Chapter 2) from lean and HFD-induced obese mice. Outcomes from this study showed that the mRNA expression of most gastric nutrient chemosensors was unchanged in HFD-induced obesity, except for a region-specific increase of antral CaSR mRNA levels. Accordingly, immunofluorescence studies explored protein expression of CaSR in the mouse stomach and co-expression with gastric ghrelin cells. The protein expression of CaSR was region-specifc with positive cells in the antrum only. Additionally, there was a high co-expression with antral ghrelin cells (≈80% colocalisation). Moreover, the density of CaSR-positive cells and co-expression with ghrelin were comparable in lean and HFD-induced obese mice. Conclusions: The stomach and gastric ghrelin cells express the cellular machinery for the detection of sweet compounds, proteins and lipids. The gastric secretion of TG and AG was modulated by proteins and lipids. However, the lipid-dependent secretion of TG and AG did not involve FFAR4 and CD36 activation. Furthermore, HFD-induced obesity did not alter the expression of most targets investigated, with the exception of an antral-specific increase in CaSR mRNA expression. However, there was no change in the density of CaSR-positive cells or the level of co-expression with ghrelin. This study provided extensive information on the nutrient-sensing ability of the mouse stomach and gastric ghrelin cells. Additional research is needed to further define the functional connections between gastric nutrient chemosensors and ghrelin secretion in health and obesity.