The effects of metformin on the gastrointestinal tract Ming Yang Robinson College, University of Cambridge This dissertation is submitted for the degree of Doctor of Philosophy September 2019 Abstract Metformin is the world’s most prescribed drug and is recommended first-line therapy for the treatment of Type 2 Diabetes Mellitus (T2DM). Numerous clinical studies have demonstrated that metformin is an effective oral agent in normalising blood glucose levels. Metformin is also one of the very few antidiabetic agents that causes modest weight loss in overweight and obese subjects. Despite its introduction into the clinic over 60 years ago, the mechanisms of metformin action are still elusive. Many of the previous studies into metformin action have focussed on its role in inhibiting endogenous glucose production in the liver, due the central role of the liver in insulin resistance and glycaemic control. However, metformin also accumulates at the highest concentrations in the small intestine, and clinical studies demonstrate that metformin formulations with low bioavailability achieve similarly effective glycaemic control compared to standard metformin formulations. These studies suggest that the gastrointestinal tract may also be an important site of metformin action. In this thesis, 2D monolayer cultures generated from intestinal organoids were used to investigate the mechanisms of metformin action in intestinal cells. Bulk RNA-sequencing and pathway analysis identified changes in glucose and amino acid metabolism pathways, as well as the upregulation of novel signalling pathways associated with the metformin response in intestinal cells. The results from RNA-seq experiments were used as the basis for functional studies into metformin action in intestinal cells. GDF-15 is a stress activated hormone that acts on its cognate receptor GFRAL expressed almost exclusively in the brainstem to elicit an inhibition of food intake. GDF-15 was one of the serum biomarkers identified in T2DM patients associated with metformin use. In a multidisciplinary collaboration, we demonstrated that Gdf15 upregulation in the distal small intestine and colon were associated with the weight loss effects of metformin. The results from transcriptomic analysis in this study identified Gdf15 as one of the most significantly upregulated genes in metformin treated intestinal cultures. Transcriptomic analysis of selected genes involved in mitochondrial function, and secretion studies reported that metformin stimulated GDF-15 secretion through mitochondrial stress and activating the integrated stress response. Previous observations from Position Emission Tomography-Computed Tomography (PET-CT) imaging in mice and T2DM patients demonstrated that oral metformin administration caused non- metabolisable fluoro-deoxyglucose (F-2DG) tracer to accumulate predominantly in the gut. This suggests that metformin increased intestinal glucose utilisation as one mechanism associated with its glucose lowering effects. In intestinal cells, metformin increased the expression of Slc2a1 (encoding i GLUT1), whilst decreasing the expression of Slc2a2 (GLUT2) and Slc5a1 (SGLT1). This was replicated in the distal small intestine of HFD-fed mice treated with metformin. Metformin also increased GLUT- transporter mediated glucose uptake in cultures. These mechanisms may potentially be mediated by AMPK and HIF1A signalling pathways. By inhibiting mitochondrial respiration, metformin also limited the metabolic plasticity of intestinal cells to utilise other metabolic fuels, which instead relied almost exclusively on glycolysis. In summary, this study suggests that increased glucose uptake mediated by GLUT transporters and increased glycolysis mediates intestinal glucose utilisation as a mechanism by which metformin causes its glucose lowering effects. Metformin may also stimulate GDF-15 secretion from the GI tract as an atypical “gut hormone”, which acts on the brainstem to reduce food intake and enhance weight loss. Together, the results from this study argue that the GI tract is an important site where metformin mediates glycaemic control and weight loss. ii Declaration This dissertation is submitted for the degree of Doctor of Philosophy. I hereby declare that this dissertation is the result of my own work and includes nothing which is the outcome of work done in collaboration with others except as specified in the main text. Marcella Ma and Brian Lam performed cDNA library preparation, read alignment and generating quantitative raw counts for bulk RNA-sequencing data as discussed in chapter 3. Pierre Larraufie performed preliminary experiments on RNA sequencing analysis. High fat diet mouse models, in vivo metformin administration, and tissue collection were performed by Tony Coll, Debra Rimmington and Irene Cimino for chapters 4 and 5. The thesis is not substantially the same as any that I have submitted, or, is being concurrently submitted for a degree or diploma or other qualification at the University of Cambridge or any other University or similar institution. I further state that no substantial part of my dissertation has already been submitted, or, is being concurrently submitted for any such degree, diploma, or other qualification at the University of Cambridge or any other University or similar institution. This dissertation does not exceed 60,000 words, excluding figures, tables, appendices and bibliography. Ming Yang, September 2019. iii Contents Abstract ........................................................................................................................................ i Declaration ................................................................................................................................. iii Abbreviations .............................................................................................................................. 1 Acknowledgements...................................................................................................................... 5 Chapter 1: Introduction ................................................................................................................ 6 1.1. Type 2 Diabetes Mellitus and Obesity ......................................................................................... 6 1.3. Treatments for obesity and T2DM ............................................................................................... 6 1.3.1. Glucose lowering agents ....................................................................................................... 6 1.3.2. Appetite regulating drugs for Obesity .................................................................................. 8 1.3.3. Bariatric surgery .................................................................................................................... 9 1.4. Metformin in the clinic............................................................................................................... 10 1.4.1. Brief history of metformin .................................................................................................. 10 1.4.2. Metformin as a glucose-lowering agent to treat T2DM ..................................................... 11 1.4.3. Metformin and the prevention of T2DM ............................................................................ 12 1.4.4. Effect of metformin on body weight ................................................................................... 13 1.4.5. Other clinical effects and applications of metformin ......................................................... 14 1.4.6. Side effects of metformin use ............................................................................................. 15 1.4.7. Genetic variants and patient responses to metformin ....................................................... 15 1.5. Background of the digestive system and the gastrointestinal tract .......................................... 17 1.5.1. The small intestinal epithelial architecture ........................................................................ 18 1.5.2. Enterocytes and nutrient absorption .................................................................................. 20 1.5.3. Intestinal nutrient metabolism ........................................................................................... 20 1.5.4. The GI tract as an endocrine organ ..................................................................................... 21 1.5.5. Intestinal organoids ............................................................................................................ 21 1.6. Mechanisms of metformin action in the liver and GI tract ....................................................... 22 1.6.1 Cellular mechanisms of action ............................................................................................. 22 1.6.2 Liver metabolism .................................................................................................................. 22 1.6.3 Intestinal glucose handling and lactate production ............................................................. 24 1.6.4 The microbiome ................................................................................................................... 25 1.6.5. Bile acid homeostasis .......................................................................................................... 25 1.6.6. GLP-1 and incretin hormones ............................................................................................