Perilipin expression in the islets of Langerhans and Surface expression in the baculovirus/insect cell system

Sammanfattning: Surface display in insect cells: Systems available for surface display that utilize bacterial phages are insufficient for displaying a number of eukaryotic proteins. By instead presenting proteins on the surface of eukaryotic cells or virus, some of these problems might be overcome. The baculovirus/insect cell system, which has a high expression rate and post-translational mashinery closely resembling the one found in mammalian cells, could possibly be used for this purpose. Earlier methods developed for surface display in the baculovirus system have been adequate when handling a single known gene but been hampered with a number of insurmountable drawbacks when handling unknown genes or complete cDNA libraries. By utilizing the signal-anchor sequence of a class II transmembrane protein some of these problems can be overcome. We have demonstrated that the signal-anchor sequence from the class II transmembrane protein neuraminidase is capable of directing proteins to all membranes, including the cell surface. Proteins fused to the signal-anchor sequence from neuraminidase are also present on the surface of budded virus. Fused proteins are displayed on the cell surface in an accessible manner, enabling selection for protein-protein interactions on a cellular level. Deletion mutants of the transmembrane region were also made in order to enhance the likelihood for successful translation of cDNAs encoding proteins in all reading frames. Perilipin in pancreatic beta-cells: Increased levels of fatty acids in the plasma, a defect associated with obesity and type II diabetes, can cause lipid deposition in non-adipose tissue and lead to toxic effects, a process termed lipotoxicity. In the islets of Langerhans, a prolonged exposure to fatty acids leads to an increased basal insulin secretion, a blunted stimulated insulin response and ultimately apoptosis. However, non-esterified fatty acids have also been shown to have a positive effect by taking part in the lipid-signaling needed for a normal glucose-stimulated insulin secretion. Perilipin is a protein known to be involved in the regulation of lipolysis and hence the release of non-esterified fatty acids in the cytosol. Perilipin has previously been detected in adipocytes and steroidogenic tissues, where it is able to both increase and decrease the release of non-esterified fatty acids and cholesterol We have now detected the expression of perilipin also in the islets of Langerhans. By overexpressing perilipin in the beta-cell line INS-1, we have demonstrated an increased resistance against lipotoxic levels of non-esterified fatty acids. Cells overexpressing perilipin had a lower level of lipolysis, despite larger triacylglycerol stores, after palmitate exposure. An increased ability to store lipids and a lower rate of lipolysis, in the cells overexpressing perilipin, preserved the stimulated insulin response and overall viability. In contrast, control cells had a completely abrogated insulin response and a decreased overall viability after exposure to the same lipotoxic levels of palmitate.