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The Role of Clostridium Bifermentans in Small Intestinal Lipid Absorption Using in Vitro and in Vivo Models

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The Role of Clostridium Bifermentans in Small Intestinal Lipid Absorption Using in Vitro and in Vivo Models The role of Clostridium bifermentans in small intestinal lipid absorption using in vitro and in vivo models Final Report by Saskia URLASS submitted at the Austrian Marshall Plan Foundation Bachelor programme: Medical and Pharmaceutical Biotechnology Internal Supervisor: Barbara Entler, Prof.(FH) Dr. External Supervisor: Kristina Martinez, PhD RD I Statutory Declaration “I declare in lieu of an oath that I have written this bachelor paper myself and that I have not used any sources or resources other than stated for its prepara- tion. I further declare that I have clearly indicated all direct and indirect quota- tions. This bachelor paper has not been submitted elsewhere for examination purposes.” Datum: 30.03.2017 Saskia Urlaß II Acknowledgment First of all, I would like to express my gratitude to my external supervisor Kristina Martinez, PhD, RD for being such a great support throughout my internship and giving me the opportunity of being part of such an exciting project. I really appreciate her patience, enthusiasm, and knowledge sharing. Moreover, I am also grateful for her help after my time in the States. I would also like to thank the principal investigator Dr. Eugene B. Chang for giving me the opportunity to work in his laboratory, and thereby giving me the possibility to gain valuable work experience in research and learning from incredible people. Ad- ditionally, I am grateful to all laboratory members, who warmly welcomed me into their team and special thanks to the administrator of the laboratory, Francine Vuko- vich for organizing my stay, including the visa and therefore making it possible for me to actually have this amazing overseas experience. I am especially thankful to the Austrian Marshall Plan Foundation for the financial support, which enabled myself to attend the internship at the University of Chicago. I would like to give my sincere thanks to my internal supervisor Dr. Barbara Entler for her great supervision and great know-how. II Abstract The worldwide obesity epidemic has tripled in the last decade and is still rising at an alarming pace, thereby afflicting millions of people with associated metabolic disor- ders, including type 2 diabetes (T2D) and cardiovascular diseases. (WHO, 2016) The gut microbiota has been implicated in playing a role in the development of obe- sity. The gastrointestinal tract (GIT) harbours 100 trillion cells of 400 species. Since the breakthrough of next-generation sequencing technology, the gut microbiota re- search made enormous progress, due to the identification of previously unknown microorganisms (MOs). (Kasubuchi et al., 2015) In addition, germ-free (GF) animal studies revealed new insights regarding the functional impact of microbes on host nutrient processing and metabolism. For example, protection against diet-induced obesity (DIO), due in part to malabsorption of fat, has been demonstrated in GF mice. (Bäckhed et al., 2007) Preliminary data from Eugene Chang’s laboratory showed that mice fed a high-fat diet have increased relative abundance of Clostridiaceae in the small intestine, de- termined by 16S rRNA sequencing. Additionally, operational taxonomic units that were significantly elevated in high-fat (HF) vs low-fat (LF) conditions shared 96% sequence similarity to Clostridium bifermentans. Therefore, my studies focused on whether or not C. bifermentans induces lipid absorption using in vitro and in vivo models. Herein, we demonstrate that conditioned media (CM) from C. bifermentans induced the expression of canonical re-esterification enzymes important for lipid absorption in small intestinal organoid cultures. C. bifermentans supplementation in vivo signif- icantly increased the expression of re-esterification enzyme Dgat2 that is especially important in triacylglycerol synthesis, and thereby contribute to increased lipid ab- sorption and transport. Taken together, our findings suggest that small intestinal microbes (i.e. C. bifermentans) are key players in lipid absorption, and that these host-microbe interactions may ultimately contribute to obesity and associated met- abolic disorders. III Table of Content Statutory Declaration ............................................................................................... II Acknowledgment ..................................................................................................... II Abstract .................................................................................................................. III Table of Content .................................................................................................... IV List of Figures and Illustrations .............................................................................. VI List of Abbreviations ............................................................................................. VII 1 Introduction ................................................................................................... 1 1.1 Obesity and gut microbiota ......................................................................... 1 1.1.1 Composition of the gut microbiota ............................................................ 1 1.1.2 Dietary impact on gut microbiota .............................................................. 4 1.2 Gut microbes and Lipid absorption ............................................................. 6 1.2.1 Small intestinal Lipid absorption ............................................................... 6 1.2.2 Gut microbiota promotes lipid absorption ................................................. 8 1.3 Clostridiaceae ........................................................................................... 10 1.3.1 Obesogenic effect of Clostridium ramosum ........................................... 10 1.3.2 Clostridium bifermentans ....................................................................... 12 1.4 Specific Aims ............................................................................................ 12 1.4.1 Aim 1 ...................................................................................................... 12 1.4.2 Aim 2 ...................................................................................................... 13 2 Materials and Methods ............................................................................... 14 2.1 Generation and Culturing of Enteroids ..................................................... 14 2.1.1 Harvesting enteroids .............................................................................. 14 2.1.2 Passaging enteroids .............................................................................. 15 2.2 Treating generated Enteroids with conditioned media (CM) ..................... 15 2.2.1 Preparation of RCM ............................................................................... 15 2.2.2 Preparation of CM .................................................................................. 16 2.2.3 Treating enteroids with conditioned media ............................................. 16 2.3 Generation and culturing of Enteroid Monolayers .................................... 20 2.3.1 Fatty Acid Uptake Assay ........................................................................ 20 2.4 Animal Study ............................................................................................ 22 2.4.1 Experimental Design .............................................................................. 22 2.4.2 Establishing growth curve for C.bifermentans ........................................ 23 IV 2.4.3 DNA extraction ....................................................................................... 25 2.4.4 Tissue collection .................................................................................... 26 3 Results & Discussion .................................................................................. 28 3.1 Conditioned media from C. bifermentans increases expression of genes involved in lipid absorption ....................................................................... 28 3.2 Enteroid Monolayer Fatty Acid Uptake Assay .......................................... 29 3.3 Animal Study ............................................................................................ 31 3.3.1 Body weight & Food intake .................................................................... 31 3.3.2 In vivo supplementation with C. bifermentans increases expression of genes involved in lipid absorption .......................................................... 32 4 Conclusion and Future outlook ................................................................... 34 List of References ................................................................................................. 36 V List of Figures and Illustrations Figure 1 Illustrates the main factors affecting microbial composition. ..................... 2 Figure 2 Illustrates the microbial development during life.. ..................................... 3 Figure 3 Illustrates the total increase in body weight measured by DEXA ............. 5 Figure 4 Illustrates lipid absorption in the small intestine ........................................ 6 Figure 5 Illustrates Triacylglycerol synthesis pathway ............................................. 7 Figure 6 Illustrates increased feacal lipid excretion of GF mice on HF diet. ............ 8 Figure 7 GF mice on HF diet are resistant to DIO and diet-induced Insulin sensitivity compared to their CONVD counterparts ................................................................
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