Taylor, Henry Adegbite (2013) Modulation of growth factor receptor trafficking as a novel approach to promoting [beta]-cell expansion. (MSc(R) thesis), Kingston University, .

Abstract

Increasing a patients' own insulin-producing beta-cell mass theoretically has the possibility of restoring glycemic control while minimizing chronic complications associated with diabetes (Shera et al. 2004). Alternatively, expanding islet beta-cells 'in vitro' could make islet transplantation more widely available, since one major limitation to this procedure is the availability of donor tissue (Efrat 2008). However, no protocol to efficiently expand adult beta-cells currently exists. The Rab family of proteins is involved in various membrane trafficking pathways. Rab7 is the key regulator of trafficking through endo-Iysosomal compartments (Stenmark and Olkkonen 2001). It directs the trafficking of proteins such as growth factor receptors from the endosomes to the lysosome for degradation instead of recycling back to the cell surface (Pereira-Leal and Seabra 2001). Rab7 down-regulation could therefore potentially be used to enhance beta-cell growth factor signaling and proliferation by increasing growth factor receptor density at the surface of the cell. In order to test this hypothesis, Rab7 siRNA oligonucleotides were transfected into the INS-1 beta-cell line and the expression level of Rab7 protein measured by western blot analysis. A range of transfection conditions was tested to achieve reproducible knockdown of Rab7 in INS-1 cells (cells that are known to be difficult to transfect), including two different transfection reagents. Using the protocol we have established, 8 western blot analysis consistently shows successful Rab7 protein reduction with a knockdown of up to 80%. We have also used confocal analysis to quantitate localization of growth factor receptors at the cell surface, rather than intracellularly (Pereira-Leal and Seabra 2001). This allowed us to test the hypothesis that Rab7 knockdown increases growth factor receptor density at the cell surface. Results showed a striking increase in staining overall for the insulin-like growth factor-1 (IGF-1) receptor (IGF-1 R), and also a higher percentage of IGF-1 R was found at the cell surface in the Rab7 small interfering ribose nucleic acid (siRNA) transfected cells in comparison to the control. In the control siRNA transfected cells, 27% of total growth factor receptors were localized to the cell membrane, while in the Rab7 transfected cells, 45% of total growth factor receptors were localized to the cell membrane. We have further shown that the activation of signaling intermediates downstream of growth factor receptors, as well as growth factor induced proliferation is enhanced in beta-cells by Rab7 knockdown. This novel data furthers the understanding of the regulation of growth and viability in islet ß-cells. If we are able to similarly enhance beta-cell growth factor responsiveness in human islet this approach could be developed in order to expand islet beta-cells either 'in vitro' or 'in vivo' to treat diabetes patients.

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