Thesis (M.Sc. (Microbiology))--Mahidol University, 1971.

Cell-based insulin therapies can potentially improve glycemic regulation in insulin dependent diabetes patients and thus help reduce secondary complications. The long-term goal of our work is to engineer autologous insulin-secreting intestinal endocrine cells as a non-beta cell approach to alleviate donor cell shortage and immune rejection issues associated with islet transplantation. These cells have been chosen for their endogenous similarity to beta cells, but generating cell constructs with sufficient insulin secretion for therapeutic effect has proven challenging. Previous work in our lab showed that a tissue engineered pancreatic substitute (TEPS) based on an engineered insulin-secreting L cell line, GLUTag-INS, was insufficient in affecting blood glucose levels in streptozotocin-induced diabetic mice, but promising since human insulin was detected in the blood. The objective of this project was therefore to fabricate an improved TEPS based on GLUTag-INS cells and evaluate its suitability as a standalone diabetes therapy. To achieve this objective, the following specific aims were (1) to investigate gene incorporation as a strategy to enhance recombinant insulin secretion from GLUTag-INS cells; (2) to develop and characterize a TEPS in vitro based on a microcapsule system containing improved GLUTag-INS cells with bioluminescence monitoring capability; and (3) to assess therapeutic efficacy of the graft in a diabetic, immune-competent mouse model and use bioluminescence monitoring to elucidate in vivo transplant behavior. This thesis therefore reports on the progression of studies from the genetic and molecular levels for improved insulin secretion per-cell, to the tissue level for enhanced secretion per-graft, and lastly to the preclinical level for therapeutic assessment in a diabetic mouse model.

Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complication associated with IDD. An autologous cell source is especially attractive for regulatory and ethical reasons and for circumventing the need for immunosuppression, which is currently standard for islet transplantation. Our approach focuses on using adult non-β-cells engineered for physiologic insulin secretion. Specifically, we utilize enteroendocrine L-cells, which naturally exhibit regulated secretion of GLP-1 in response to physiologic stimuli, and upon genetic engineering, co-secrete insulin in a regulated manner. The overall goal of this project is to develop a tissue engineered pancreatic substitute based on a recombinant enteroendocrine cell line and test the efficacy of the pancreatic substitute by implantation into diabetic mice. The specific aims of this thesis were to (1) to modify murine L-cells for regulated insulin secretion and evaluate the insulin secretion properties of the recombinant cells; (2) to incorporate insulin-secreting L-cells into an implantable construct containing small intestinal submucosa (SIS) and to evaluate insulin secretion from the construct in vitro; and (3) to test the efficacy of the tissue engineered pancreatic substitute in vivo by implanting it intraperitoneally in mice made diabetic by streptozotocin. Thus, this proposal takes a tissue engineered pancreatic substitute for IDD from in vitro development to in vivo testing.

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Type 2 diabetes (T2D) is a chronic metabolic disease characterized by hyperglycemia, insulin resistance, and the dysfunction of β-cells. While there are several therapies for T2D, there is no effective treatment that can reverse the functional decline of pancreatic β cells in T2D patients. Glucagon-like peptide-1 (GLP-1) is a peptide hormone secreted by human intestinal L cells, which can stimulate the proliferation and differentiation of β cells and promote glucose-stimulated insulin secretion (GSIS), thereby playing a critical role in maintaining glycemic homeostasis. Recently, GLP-1-based medications have been developed for treating T2D. However, most of the GLP-1-based drugs are expensive and have significant adverse effects. Therefore, development of safer and more convenient agents that can mimic the physiologically fed state to promote endogenous GLP-1 secretory function of intestinal L-cells to improve glucose homeostasis holds great potential for the prevention and treatment of T2D. This project aimed to examine whether natural compound genipin promotes intestinal GLP-1 secretion and exerts anti-diabetic effects. I found that genipin rapidly increased GLP-1 secretion from intestinal L-cells, with 10 and 100 μM concentration inducing significant incretin hormone release. L-cells exposed to genipin displayed a rapid increase in intracellular [Ca²⁺]i and the activity of phospholipase C (PLC). Inhibition of PLC ablated genipin-stimulated Ca²⁺] increase and GLP-1 secretion, suggesting that genipin-induced GLP-1 release from the cells depends on the PLC/Ca²⁺ pathway. In vivo, genipin reduced the non-fasting and fasting blood glucose levels, improved insulin resistance, and protected again high fat diet-induced liver damage. All together, these data indicate that genipin is a naturally occurring anti-diabetic agent, which could be a pharmaceutical lead for developing anti-diabetic drugs. / M.S. / More than 34 million Americans are suffering from diabetes, with over 90% of these cases being type 2 diabetes (T2D). Loss of β-cell mass and function is central to the deterioration of glycemic control over time in T2D. Therefore, preservation or improvement of β-cell mass and its insulin secretory function could prevent and treat T2D. While there are several pharmaceutical therapies for T2D, no effective treatment is available for reversing functional decline of pancreatic β-cells in T2D patients. It has been well recognized that glucagon-like peptide-1 (GLP-1), which is an incretin hormone secreted from intestinal L-cells, plays a critical role in maintaining glycemic homeostasis via potentiating glucose-stimulated insulin secretion and promoting β-cell proliferation. This present work is to determine whether natural compound genipin promotes intestinal GLP-1 secretion and thus exerts anti-diabetic effect.

<p>As obesity has become one of the most prevalent metabolic diseases, and diabetes mellitus has become the seventh leading causes of death in the United States, alternative food/nutrition-based approaches to tackle obesity that are both efficacious and cost effective are in high demand. Since starch and its derived products are the principal dietary supply of glucose, strategies of using slowly digestible starch to achieve moderated glycemic response and prolonged glucose delivery, as well as to locationally digest starch into the ileum, have shown successful results such as moderation of insulinemia and reducing food intake in obese animals. An important regulator of appetite suppression is the neuroendocrine system of the gut-brain axis. Glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), and peptide YY (PYY) are the main anorexigenic peptide products of the intestinal enterendocrine L-cells that regulate postprandial insulin levels as well as satiety signals. The stimulation of the enteroendocrine L-cells throughout the gastrointestinal tract through glucose, fatty acids and proteins has been extensively studied and confirmed. However, the stimulatory effect of complex dietary carbohydrates on L-cells is not described. In this dissertation, we investigated the <i>in vitro</i> intestinal cell chemosensation of L-cells to α-amylase starch digestion products, named maltooligosaccharides (MOS), and in the possible application of using slowly digestible starch delivery of MOS <i>in vivo</i>.</p> <p>In Chapter II of this dissertation, we reported a significantly higher stimulatory effect of MOS on GLP-1 and OXM secretion compared to glucose in mouse and human L-cells, respectively. Additionally, maltotriose enhanced the relative expression of the gastrointestinal peptide, cholecystokinin. Moreover, MOS exhibited protective effects on barrier function and monolayer integrity of intestinal epithelial cells. </p> <p>In Chapters III and IV, we performed a multiomics approach where transcriptomic analysis and global protein profiling of mouse L-cells treated with different types of MOS showed that the carbohydrates exhibit their effects through the induction of exocytosis of GLP-1- or OXM-containing vesicles and not through a positive regulation of the proglucagon gene expression. It is suggested that MOS induce higher secretion, but not higher synthesis, of the proglucagon gene products. In addition, maltotriose treatment downregulated the relative expression of the glucotoxicity marker, thioredoxin-interacting protein, and upregulated the relative expression of tight junction proteins supporting a role of MOS in barrier function integrity.</p> <p>Translating the <i>in vitro</i> findings into an <i>in vivo</i> application that is beneficial for human health required the use of controllable tool for the delivery of MOS throughout the small intestine for sensing by a higher number of L-cells. Slowly digestible starch (SDS), compared to rapidly digestible starch, provided such a tool. For this purpose, we used alginate-entrapped SDS microspheres that digest distally into the ileum to examine the role of SDS in the intervention and prevention of obesity in C57BL/6J diet-induced obese (DIO) and lean mice models.</p> <p>Results showed that 20% SDS in low-fat diets significantly improved weight loss and food intake reduction in DIO mice converted to low-fat diet for 12 weeks. Similarly, 15% SDS in high-fat diets showed significant reduction in body fat percent and significant increase in lean body mass as well as considerable reduction in weight gain rate and food intake in lean mice fed on 45% of calories high-fat diet. Immunohistochemistry of small intestine of mice in both the intervention and prevention studies revealed an even and thorough distribution of GLP-1 positive L-cells.</p> <p>Overall, this dissertation proposes several insights into L-cell sensation of dietary starch-degraded MOS delivered by the consumption of slowly digestible starch. MOS exhibit unique influences on L-cell sensitivity and gut hormone productivity. Future research investigating the mechanisms of intestinal sensing of MOS, as well as the development of bioactive carbohydrate structures that could preserve body weight and modulate glucose tolerance <i>in vivo</i> is needed to translate these findings into nutritional recommendations and food products beneficial for human health. The intricate role of dietary carbohydrates on gut physiological response, related to satiety and food intake could be a new approach for design of foods for health applications.</p>

Scope and Contents: Virus specific RNA components of the cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis virus were examined. Studies were carried out both in the presence and absence of defective particle interference. / Thesis / Master of Science (MSc)

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Previous research of colonies formed by yeast Saccharomyces cerevisiae growing on glycerol agar medium revealed two major cell types of U and L cells that are formed within these colonies. This colonial cell differentiation seem to be caused by communication among yeast cells as well as whole colonies and affected by changes in the environment (for example changes in nutrients). Studies of U and L cells showed that U cells are more resistant against biological, chemical and physical stresses than L cells. The aim of this thesis was to isolate U and L cell types and investigate their resistance against selected low molecular weight chemical substances produced in Ehrlich pathway. Ehrlich pathway was discovered in 1907 and was classified as amino acid catabolic pathway in yeast S. cerevisiae. The low molecular intermediates are formed in Ehrlich pathway which are called fusel (original name from German) alcohols and acids. These chemical substances are widely used in food industry and cosmetics especially because of their aroma. Several studies provided indications that these chemical substances may affect development of colonies and important yeast functions such as switching to the pseudohypfal growth of S. cerevisiae cells. Some chemical substances of the Ehrlich pathway were selected and their effects on...