75 research outputs found
Recommended from our members
Mixed primary cultures of murine small intestine intended for the study of gut hormone secretion and live cell imaging of enteroendocrine cells
The gut is the largest endocrine organ of the body, with hormone-secreting enteroendocrine cells located along the length of the gastrointestinal epithelium. Despite their physiological importance, enteroendocrine cells represent only a small fraction of the epithelial cell population and in the past, their characterization has presented a considerable challenge resulting in a reliance on cell line models. Here, we provide a detailed protocol for the isolation and culture of mixed murine small intestinal cells. These primary cultures have been used to identify the signaling pathways underlying the stimulation and inhibition of gut peptide secretion in response to a number of nutrients and neuropeptides as well as pharmacological agents. Furthermore, in combination with the use of transgenic fluorescent reporter mice, we have demonstrated that these primary cultures become a powerful tool for the examination of fluorescently-tagged enteroendocrine cells at the intracellular level, using methods such as patch clamping and single-cell calcium and cAMP-FRET imaging.GLP-1 immuno-assays were performed by the Core Biochemical Assay Laboratory at Addenbrookeâs Hospital. This work has, over the years, been supported by the Wellcome Trust (currently active grants 106262/Z/14/Z and 106263/Z/14/Z) and the Medical Research Council (MRC) (grants MRC_MC_UU_12012/3 and MRC_MC_UU_12012/5)
Recommended from our members
Chylomicrons stimulate incretin secretion in mouse and human cells
Aims/hypothesis Lipids are a potent stimulus for the secretion of glucagon-like peptide (GLP)-1 and glucose-dependent insulinotropic peptide (GIP). Traditionally, this effect was thought to involve the sensing of lipid digestion products by free fatty acid receptor 1 (FFA1) and G-protein coupled receptor 119 (GPR119) on the apical surface of enteroendocrine cells. However, recent evidence suggests that lipids may in fact be sensed basolaterally, and that fatty acid absorption and chylomicron synthesis may be a prerequisite for their stimulatory effect on gut peptide release. Therefore, we investigated the effect of chylomicrons on GLP-1 and GIP secretion in vitro.
Methods The effect of chylomicrons on incretin secretion was investigated using GLUTag cells and duodenal cultures of both murine and human origin. The role of lipoprotein lipase (LPL) and FFA1 in GLUTag cells was assessed by pharmacological inhibition and small (short) interfering RNA (siRNA)-mediated knockdown. The effect of chylomicrons on intracellular calcium concentration ([Ca2+]i) was determined by imaging GLUTag cells loaded with Fura-2. In the primary setting, the contributions of FFA1 and GPR119 were investigated using L cell-specific Gpr119 knockout cultures treated with the FFA1 antagonist GW1100.
Results Chylomicrons stimulated GLP-1 release from GLUTag cells, and both GLP-1 and GIP secretion from human and murine duodenal cultures. Chylomicron-triggered GLP-1 secretion from GLUTag cells was largely abolished following lipase inhibition with orlistat or siRNA-mediated knockdown of Lpl. In GLUTag cells, both GW1100 and siRNA-mediated Ffar1 knockdown reduced GLP-1 secretion in response to chylomicrons, and, consistent with FFA1 Gq-coupling, chylomicrons triggered an increase in [Ca2+]i. However, LPL and FFA1 inhibition had no significant effect on chylomicron-mediated incretin secretion in murine cultures. Furthermore, the loss of GPR119 had no impact on GLP-1 secretion in response to chylomicrons, even in the presence of GW1100.
Conclusions/interpretation Chylomicrons stimulate incretin hormone secretion from GLUTag cells as well as from human and murine duodenal cultures. In GLUTag cells, the molecular pathway was found to involve LPL-mediated lipolysis, leading to the release of lipid species that activated FFA1 and elevated intracellular calcium.FMG and FR are funded by grants from the Medical Research
Council (MRC_MC_UU_12012/3 and MRC_MC_UU_12012/5) and
Wellcome Trust (grants 106262/Z/14/Z and 106263/Z/14/Z). This work
was also supported by a Society for Endocrinology Early Career Grant
(AP
Complementary intestinal mucosa and microbiota responses to caloric restriction
The intestine is key for nutrient absorption and for interactions between the microbiota and its host. Therefore, the intestinal response to caloric restriction (CR) is thought to be more complex than that of any other organ. Submitting mice to 25% CR during 14 days induced a polarization of duodenum mucosa cell gene expression characterised by upregulation, and downregulation of the metabolic and immune/inflammatory pathways, respectively. The HNF, PPAR, STAT, and IRF families of transcription factors, particularly the Pparα and Isgf3 genes, were identified as potentially critical players in these processes. The impact of CR on metabolic genes in intestinal mucosa was mimicked by inhibition of the mTOR pathway. Furthermore, multiple duodenum and faecal metabolites were altered in CR mice. These changes were dependent on microbiota and their magnitude corresponded to microbial density. Further experiments using mice with depleted gut bacteria and CR-specific microbiota transfer showed that the gene expression polarization observed in the mucosa of CR mice is independent of the microbiota and its metabolites. The holistic interdisciplinary approach that we applied allowed us to characterize various regulatory aspects of the host and microbiota response to CR
Randomised clinical study: inulin short-chain fatty acid esters for targeted delivery of short-chain fatty acids to the human colon
SUMMARY Background Short-chain fatty acids (SCFA) produced through fermentation of nondigestible carbohydrates by the gut microbiota are associated with positive metabolic effects. However, well-controlled trials are limited in humans
De novo assembly of genomes from long sequence reads reveals uncharted territories of Propionibacterium freudenreichii
New insight into inter-organ crosstalk contributing to the pathogenesis of non-alcoholic fatty liver disease (NAFLD)
Flavone Hispidulin Stimulates GlucagonâLike Peptideâ1 Secretion and Ameliorates Hyperglycemia in StreptozotocinâInduced Diabetic Mice
Mixed Primary Cultures of Murine Small Intestine Intended for the Study of Gut Hormone Secretion and Live Cell Imaging of Enteroendocrine Cells
The short chain fatty acid propionate stimulates GLP-1 and PYY secretion via free fatty acid receptor 2 in rodents.
- âŠ