Abstract

Our previous work has shown that an allosteric relationship exists between circulatory NEFA transport and plasma zinc handling through binding to HSA. This has led to our core hypothesis that plasma zinc speciation is influenced by NEFAs in disease states and impacts upon processes including coagulation and insulin signalling. We will examine how binding of complex (patho)physiologically relevant mixtures of NEFAs alter Zn2+ binding to HSA using ITC. This work is based upon our previous measurement of NEFAs in type-II diabetes patients and controls. We will examine how NEFAs influence plasma zinc speciation using an established approach incorporating 2D-PAGE combined with LA-ICP-MS, which will be further developed to include bidimensional separation techniques. We will also develop a bespoke approach incorporating bidimensional separation with quantitative MS and ICP-MS to assess NEFA-induced speciation changes. The zinc-binding properties of interesting proteins identified will be probed using ITC, ESI-MS and IMMS analyses. The effect of NEFA-induced changes in the zinc-binding properties of HSA on insulin hexamer breakdown will be assessed using a FRET-based approach utilising Cy3- and Cy5-labelled insulin. Zinc flux in primary HUVECs will be assessed using our previously established stable Zn isotope-based approach to determine how NEFAs influence cellular zinc homeostasis, with intracellular speciation examined using the 2D-gel electrophoresis/LA-ICP-MS approach detailed above. Combining these two approaches will enable us to identify proteins that load with Zn2+ as influx increases in response to NEFAs. Finally, we will examine how NEFAs influence insulin signalling in HUVECs using molecular and gene expression-based approaches. Activation of PI3K/AKT/eNOS and Ras/Raf/MEK pathways will be assessed using specific antibodies to active forms of these proteins and insulin-dependent gene expression will be examined using a Stat5-dependent luciferase assay.