Mechanisms underlying monosaccharide stimulated incretin hormone secretion from the intestine

Abstract

Obesity and type II diabetes (T2D) are healthcare crises with continually rising prevalence worldwide. Drugs which mimic the gut hormone glucagon-like peptide-1 (GLP-1), e.g. Semaglutide, are currently the most effective obesity and T2D therapeutics, activating GLP-1 receptors in the pancreas and brain to improve blood glucose and appetite control. Due to the therapeutic benefits of GLP-1 signalling, understanding the factors that regulate its release are of continued interest. Gut hormones are produced by enteroendocrine cells (EECs) of the intestinal epithelium and are released in response to a variety of intestinal signals, with diverse roles in the control of metabolism. The gut hormones GLP-1 and glucose-dependent insulinotropic polypeptide (GIP), known as incretins due to their insulinotropic properties, are produced and released by the EEC sub types L-cells and K-cells respectively. The monosaccharide glucose is a trigger for incretin secretion, which requires glucose uptake via the sodium-dependent glucose transporter 1 (SGLT1) expressed by L-cells and K-cells, as demonstrated by in vitro cell based studies. SGLT1 is expressed throughout the intestinal epithelium and additionally facilitates glucose absorption to the bloodstream. In contrast to in vitro experiments, in vivo studies where humans or animal models lack functional SGLT1, show that GLP-1 is still secreted in response to oral glucose, while GIP secretion is absent. In this thesis, we aimed to explore potential SGLT1 independent mechanisms by which the presence of unabsorbed glucose in the intestine triggers GLP-1, but not GIP, secretion in vivo. We first confirmed that an oral glucose challenge resulted in GLP-1, but not GIP secretion in Sglt1 knock out (Sglt1-/-) mice, while the secretion of both hormones was observed in wild-type mice. We then showed that SGLT1 substrates (α-MDG (α-Methyl-D-Glucose), D-Glucose) stimulated GLP-1 secretion from ileal organoids, but that this GLP-1 secretion was absent in experiments using Sglt1-/- ileal organoids. These experiments confirmed the requirement of SGLT1 for in vitro, but not in vivo, glucose-stimulated GLP-1 secretion. Using metabolomic techniques we subsequently determined that fermentation of unabsorbed glucose by the gut microbiome generated short chain fatty acids (SCFAs) in the colon of Sglt1-/- mice, which have the potential to stimulate GLP-1 release. However, through studies using a panel of glucose analogues which are not microbiome substrates (3-OMG (3-O-Methyl-D-Glucose), L-Glucose), antibiotic mediated microbially depleted Sglt1-/- mice and metabolomic techniques, we demonstrated that the microbiota and SCFA synthesis were not essential for SGLT1 independent glucose-stimulated GLP-1 release. We observed that GLP-1 secretion triggered by an unabsorbable intestinal glucose load occurs alongside intestinal distension in both microbially intact and depleted mice, which may exert pressure on the intestinal epithelium. To explore whether increased intra-intestinal pressure might trigger GLP-1 release from L-cells, we initially used RNAsequencing techniques to generate transcriptomic datasets of EECs. These datasets facilitated the investigation of EEC expression of “mechanosensors”, which could convert mechanical stimuli, such as pressure, to hormone release. We described expression of the mechanosensitive G protein-coupled receptor AT1aR (angiotensin II receptor type 1a) and the mechanosensitive ion channel Piezo2 by L-cells, but not K-cells, presenting potential mechanisms by which an unabsorbed glucose load in the intestine may trigger GLP-1, but not GIP, secretion in vivo. Investigations into the functional relevance of mechanosensors expressed by L-cells may lead to pharmacological targets which could be explored for the development of novel therapeutics to treat obesity and T2D by stimulating endogenous GLP-1 release from the intestine.