Characterising the role of insulin receptors in adipogenesis

Insulin possesses the ability to promote a wide range of cellular responses with links to disease states such as diabetes, cancer, and obesity. The receptors for insulin are numerous, arising from alternative splicing of the INSR gene and heterodimerisation of insulin receptor (IR) isoforms and insulin-like growth factor 1 receptor (IGF-1R) subunits. Whether the different cellular responses to insulin are attributable to particular receptors has not been well defined; the aim of this investigation was to elucidate this uncertainty in the context of adipogenesis. Using a human mesenchymal stem cell line (Y201 MSCs) as an alternative to the widely-used murine 3T3-L1 fibroblasts, the suitability of these cells was first determined prior to their application in the investigation of insulin signalling and adipogenesis; proteomic and functional characterisation revealed Y201 MSCs to be comparable to 3T3-L1 fibroblasts for this purpose. Analysis of the changes in expression of INSR and IGF-1R and differences in the abundance of IR:IGF-1R heterodimers provided concordant results reflecting the major roles of these proteins and revealed unexpected patterns in heterodimer formation for undifferentiated and differentiated cells following insulin stimulation. A potential explanation for these patterns was surmised but remains to be validated experimentally. Furthermore, the CRISPR-Cas9-directed generation of INSR-/- and IGF-1R-/- cell lines was performed but additional screening is required to confirm successful gene knockout. Finally, constructs enabling the expression of a double-tagged GLUT4 construct were generated to allow future analysis of the metabolic signalling capacity of each receptor. In summary, this work successfully characterised an alternative cell line with respect to adipogenesis and identified changes in IR and IGF-1R expression throughout this process; more work must be done to directly investigate the effect of each of these receptors in this process and the foundations for necessary future experimentation have been laid in this project.