Gene expression profiling of in vitro-generated pancreatic islet cells (Peck, Recordi, She)

Type 1 diabetes is due to destruction of the insulin-producing beta cells by one’s own immune system. Islet cell transplantation is a potential cure for the disease if one can prevent the recurrent autoimmune attack on transplanted islet cells and sufficient islet cell supplies can be obtained. The investigators at UF and UM are tackling these problems from multiple fronts, which can benefit from gene expression profiling. We will focus on the islet cells cultured in vitro here. During embryogenesis, mature functional islets develop from a pool of undifferentiated stem cells associated with pancreatic ductal epithelium. Recently, the technology for isolation and propagation of islet-producing stem cells (IPSCs) that give rise to islet progenitor cells (IPCs) capable of differentiating in vitro to mature, functional islets containing ?-, ?- and ?-cells in mice and humans has been perfected in Dr. Peck’s laboratory. The ability to control growth and differentiation of IPSCs and IPCs through the various stages of islet development offers a unique model to study temporal changes in gene expression associated with islet development, as well as to define cell surface markers expressed by IPSCs, IPCs and islet cells, in addition to factors important for organogenesis of the endocrine pancreas. We have begun to apply cDNA microarray analysis on distinct cell populations isolated from established IPSC / IPC lines and in vitro-generated islets in order to identify temporal changes in gene expression(s) during growth and differentiation of pancreatic endocrine cells. This approach will identify a large number of candidate genes that will, subsequently, permit the isolation of full-length DNA sequences for the differentially expressed genes as well as the determination of their precise expression patterns during differentiation of endocrine pancreas into mature, functional islet cells. Determining the role of genes and their corresponding gene products in controlling growth and differentiation of endocrine pancreas will facilitate production of reagents that identify subpopulations of islet cells and may provide insight into regulation of islet development. Gene expression analyses will enable the identification of genes that control the stepwise differentiation of endocrine pancreatic stem cells to ?-, ?- and ?-cells. In addition, screening of genes expressed at various stages of growth and differentiation will permit identification of islet cell growth factors, gene products regulating islet development, and unique differentiation markers for IPSCs and IPCs. By understanding islet stem cell growth and differentiation, we may be able to generate in vitro sufficient numbers of functionally active, yet "immunologically acceptable" islets for successful implantation and treatment of type 1 diabetes and some day regulate the growth of pancreatic islets in vivo directly from ductal epithelia. Project 5. Response to glucose deprivation in 3T3-L1 adipocytes (Frost and Laipis): In mammalian systems, carbohydrates function not only as substrates for growth but as effectors of sugar sensing systems that initiate changes in gene expression. Both abundance and depletion of carbohydrates can enhance or repress the expression of genes. Carbohydrate-regulated genes provide a mechanism for integrating cellular response with resource utilization in the entire body. This coordinated effort provides exquisite regulation in maintaining the circulating levels of glucose in humans within a fairly narrow range. Glucose deprivation increases the rate of glucose transport in a number of cell types by a protein synthesis-dependent mechanism. Relevance to the human system is seen clearly in the adipocyte model system, 3T3-L1 adipocytes, where the rate of transport fluctuates about 4-fold between hyperglycemic (25mM) to hypoglycemic (3mM) conditions after 12h of treatment, levels which are within the circulating range in humans. Complete deprivation can be used in cell culture to maximize the effect. In a number of cell types, an increase in the expression of GLUT1, the constitutive glucose transport, accounts for the increase in transport activity. However, in the adipocyte model system, neither the GLUT1 message abundance nor protein levels change. This proposal focuses on determining the mechanism by which transport activity increases. The studies will test the hypothesis that a novel protein is produced in response to glucose deprivation, which in turn alters the activity of GLUT1. An alternative hypothesis includes the production of a new isoform of the glucose transporter family in response to glucose deprivation, analogous to yeast where multiple glucose transporter proteins function to optimize glucose utilization. Regardless, 3T3-L1 adipocytes, which are of mouse origin, will be exposed to medium with (the reference state) or without 25mM glucose (the test state) for 12h which maximizes the response to glucose deprivation. From previous experience, we know that from each 10cm plate of cells we will be able to isolate about 80?g of total RNA. Microarray analysis should identify a number of gene expression changes. Candidate genes which encode for activators or transporters must show substantial change in expression to be of interest as transport activity fluctuates some 20-fold. We will determine the full length cDNA sequence for such new genes of interest. The cloned gene(s) will be inserted into an adeno-associated virus vector for overexpression in control 3T3-L1 cells to determine ability to increase transport activity. Identification of the protein(s) which increases transport activity, whether it be an activator or a transporter, could provide future means by which to control hyperglycemia in both insulin-dependent and -independent diabetes.
 

Visit our Site Map to see what is available or use the navigation menu to the left.