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Functional analysis of transcription factors in L-cell biology

Final Report Summary - TRAFALOGY (Functional analysis of transcription factors in L-cell biology)

Executive Summary

Worldwide, close to 2 billion people are overweight, obese or suffer from diabetes according to the World Health Organisation. When energy intake (by consumption of food) is chronically higher than energy expenditure (by producing heat and physical activity), the surplus energy is stored in adipose tissue, i.e. fat, which will finally result in obesity. Obesity by itself is a risk factor for cardiovascular disease, but also diabetes. In healthy people, the hormone insulin maintains blood sugar levels in a physiological range by acting on several tissues, including liver and muscle. In obese patients, the ability of insulin to reduce blood sugar levels is reduced (insulin resistance). When obesity persists, insufficient amounts of insulin are produced, and blood sugar levels will eventually be constantly too high – the outbreak of diabetes. No cure is available for obesity and diabetes, and current treatments work only in a part of the population, or gradually lose their efficacy over time. Recently, the ability of several gut hormones including glucagon-like peptide 1 (GLP1) to control body weight and blood glucose levels were identified. GLP1 and other hormones are secreted from specialised intestine cells (L-cells) upon intake of a meal, and act on several tissues including the brain and the pancreas to signal incoming energy (as the meal is digested). These hormones and their functions are conserved in humans, and long-lasting GLP1-like therapeutics are already being used for the treatment of diabetes. Nonetheless, little is known about the cells that secrete GLP1, the L-cells. In this project, the gene expression of immortalized mouse and human immortalized L-cells were compared, as were all secreted and cleaved proteins from these cells. An in vitro screen was performed to define potential regulators of L-cell mitosis, apoptosis, differentiation and GLP1 secretion, which led to the identification of several potential regulators of these processes. Finally, the molecular mechanisms controlled by several of these candidate genes was defined by biochemical and genetic analysis in vitro. Taken together, these insights into L-cell biology increase our understanding of L-Cells, which might assist in developing novel therapeutic angles to treat obesity and diabetes.
Summary description of project context and objectives
Incidence of obesity and diabetes has reached pandemic proportions, with 38% of the adult population being overweight, and close to 10% suffering from diabetes (WHO data, 2014). Alarmingly, the rate of childhood obesity increases as well. Obesity is characterized by a deregulation between energy intake and energy expenditure, causing increased deposition of energy in fat. Obesity is linked to decreasing cellular sensitivity to insulin, a hormone secreted by pancreatic beta-cells, that maintains normal blood glucose and lipid levels. While pancreatic beta-cells are able to increase insulin output at the beginning, when chronic obesity occurs, eventually insulin secretion is insufficient to keep blood glucose and lipid levels in a physiological range, and diabetes mellitus occurs. Diabetes itself is life-threatening and a leading cause for blindness, loss of kidney function, impaired wound healing, cardiovascular disease and a risk factor for dementia. No cure for obesity and diabetes is available, and current therapeutics have considerable side effects, or are only effective in parts of the population, or slowly lose their efficacy over time. Body weight and energy metabolism is controlled by a complex hormonal system in mammals, and recently, the key role of intestinal hormones has come into focus of research. In the intestine, specialized cells sense food intake by direct and indirect mechanisms, for example by extension of the gut, by fatty acid receptors or neuronal input onto the cells. In response, these cells known as L-cells secrete several hormones such as glucagon-like peptide 1 (GLP1), oxyntomodulin (OXM) or peptide tyrosine tyrosine (PYY). These hormones in turn act on multiple target tissues such as brain, liver and pancreas and thereby regulate food intake, glucose production, insulin secretion and intestinal contraction, among others. Among these hormones, GLP1 is able to reduce blood glucose concentrations and body weight, and GLP1-like agonists or inhibitors of GLP1-degrading enzymes are currently used as treatments for diabetes. On the other hand, the molecular pathways controlling synthesis and secretion of GLP1 and other L-cell hormones are not well described. In this regard, several immortalized cell lines are currently used to test scientific hypotheses, for example murine STC1 cells.
This project aimed to increase our understanding of the biological processes behind mitosis, apoptosis, and hormone secretion from L-cells. To this end, gene expression of immortalized L-cells was to be compared with gene expression of primary L-cells derived from transgenic mice expressing a glucagon promoter-driven fluorescent protein. 50 candidate genes expressed at high levels in L-cells were to be screened for their ability to alter either survival or mitosis of L-cells, and/or to impact on secretion of hormones, such as GLP1 itself. The most promising candidate genes involved in L-cell biology in vitro were to be characterized in further in vitro experiments, including overexpression experiments.

Description of main results
To gain insight into critical mediators of L-cell function, we used mass-spectrometry to assess N-linked glycoproteins as well as shedded and secreted proteins from murine and human L-cell lines using established methods, and identified close to 900 proteins either secreted or shedded from these cells. These included (among others) proteases, receptors, hydrolases, cell adhesion molecules and putative transcription factors. Since previous reports have underlined the importance for proteases in the regulation of endocrine cells, we concentrated on a subset of 30+ proteases found in our screen. To establish the biological function of these candidate genes, we first established GLP1 secretion assays under multiple conditions in L-cell lines, such as with and without fatty acid-stimulation. These experiments verified the ability of fatty acids to stimulate GLP1 secretion in vitro. We next performed knockdown of the candidate genes in STC1 cells by transfection of siRNA pools, and measured GLP1 secretion, glucagon expression and cell viability. These screens revealed several candidate proteins that alter glucagon expression and GLP1 secretion without affecting cell survival or mitosis in vitro. For several candidate genes, siRNAs with highly efficacious knockdown were determined and the assays as mentioned above, repeated. Moreover, we investigated tissue-specific expression of candidate genes by performing gene-specific quantitative PCR in multiple mouse tissues. To directly examine if candidate genes are expressed in primary L-cells, we performed immunohistochemistry for multiple candidate genes in intestinal sections and co-stained for GLP1. For some candidate genes, overlapping signals were detectable, indicating co-expression in L-cells. For several candidate genes, overexpression studies were performed to gain insight into their cellular function.
To gain insight in the molecular mechanisms controlling primary L-cell function, we generated transgenic mice expressing green fluorescent protein in L-cells, and established protocols to separate (green) L-cells from non-fluorescent intestinal cells from both small and large intestine. We performed RNA-sequencing of primary and L-cell lines. We used these RNA-sequencing profiles in combination with immunohistochemistry using specific antibodies against the candidate genes to directly assess if candidate genes are detectable in primary L-cells. Moreover, we used bioinformatics approaches to compare expression profiles of L-cell lines and primary L-cells, and found that while key genes involved in L-cell hormone secretion were also expressed at high levels in L-cell lines, several hormones not expressed in primary L-cells were detectable in L-cell lines, indicating that some differential expression between primary L-cells and L-cell lines exist.

Potential impact
Intestinal biology has moved into the focus of current research interest, as the interaction between intestinal gut bacteria, gut hormones and metabolism is becoming increasingly clear. The ability of gut hormones such as GLP1, Oxyntomodulin and PYY to act beneficially on systemic metabolism is conserved between mice and man, and indeed GLP1 analogues and inhibitors of GLP1 degrading enzymes are available as diabetes therapy. Surprisingly, much less is known about L-cells, the cells that secrete these hormones. In the current project, we have shed light on crucial biological processes taking place in these cells by using state-of-the-art genetic tools in vivo and in vitro. In detail, we have defined the sheddome and secretome of L-cell lines as well as the transcriptome of primary L-cells. Several genes essential for normal GLP1 secretion in L-cell lines in vitro were detected and molecularly defined. Secreted and shedded proteins have been shown to be crucially involved in controlling biological processes in other endocrine cell types, such as beta cells. Both secretion and shedding of these proteins may be pharmacologically targeted to influence metabolism, and the data gained by these experiments may help to define critical pathways affecting function and viability of these metabolically important cells. In this regard, future in vivo experiments must be performed to directly test the involvement of the candidate genes in GLP1, oxyntomodulin and PYY secretion and general L-cell biology in vivo.