- Title
- The role of Zinc Transporters in modulating Insulin signalling
- Creator
- Nield, Alex
- Date
- 2015
- Type
- Text; Thesis; PhD
- Identifier
- http://researchonline.federation.edu.au/vital/access/HandleResolver/1959.17/99999
- Identifier
- vital:10458
- Identifier
- https://library.federation.edu.au/record=b2713003
- Abstract
- Zinc is a cell impermeable transition metal with a large number of biological functions, and is an essential component of the insulin signalling pathway. Cellular free zinc increases insulin sensitivity though it is also toxic at high levels, making it essential for cells to tightly regulate bioavailable levels. This homeostasis is maintained by three groups of proteins known as Zips, ZnTs and metallothioneins (MTs). Zips and ZnTs are zinc transporters with Zips increasing cytosolic zinc by pumping it outside the cell or from organelle stores, while the ZnTs decrease cytosolic zinc. The MTs bind to free zinc in the cytosol, reducing its bioavailability. The rapid release of zinc mediated by these proteins has been implicated as a mechanism of signal pathway activation, through zinc activating and deactivating various signalling proteins. This thesis investigated one zinc transporter in particular, known as Zip7. Zip7 is a novel Zip transporter localised to the endoplasmic reticulum and has been implicated in cell signalling in breast cancer cells through release of zinc from cellular stores in response to extracellular stimuli. The aim of this thesis was to investigate the potential role of this zinc transporter in modulating the insulin signalling pathway. The human Zip7 protein sequence was analysed using various bioinformatics tools to identify regions that may contribute to the proposed novel function of this transporter. The loop regions of Zip7 were found to be poorly conserved between species with the exception of histidine rich regions, which showed a high level of conservation when compared to a diverse series of species and so are suspected to have an essential role in modulating the transport function of Zip7 by binding to zinc. These findings implicate histidine residues as an important functional component of Zip7. In order to identify whether Zip7 expression is essential for a normal insulin response, Zip7 mRNA was reduced via transfection of siRNA in mouse skeletal muscle cells and measurement of markers of insulin signalling. When Zip7 expression was reduced there was a subsequent decrease in the expression of several markers of insulin signalling including Glut4 protein levels, Akt phosphorylation and insulin-mediated glycogen synthesis, indicating that the cells were insulin resistant compared to the control. It was hypothesised that given the proposed role for Zip7 in mediating rapid zinc release and that Zip7 expression is important for normal insulin signalling, Zip7 activation is stimulated by insulin treatment to temporarily increase cytosolic zinc bioavailability as a positive feedback mechanism for prolonging pathway activation. To test this, live cell imaging of zinc flux in cells was performed in cells with reduced Zip7 expression compared to controls. Insulin was shown to cause an increase in cytosolic zinc in C2C12 cells. However when Zip7 expression was reduced, even though the cells showed signs of insulin resistance, there was still an increase in zinc levels mediated by insulin. Insulin treatment is known to induce cellular ROS production and hydrogen peroxide has been suggested to cause a release of zinc due to oxidation of MTs leading to a release of bound zinc. These findings indicate that insulin-stimulated zinc flux is the result of MT oxidation rather than Zip7 activation. Taken together, these results highlight an important role for Zip7 in the insulin signalling pathway and show a previously undescribed positive feedback loop whereby insulin mediates a release of zinc to potentially inhibit PTP1B and other phosphatases to prolong insulin signalling activation. Further work is needed to fully elucidate the role of Zip7 in this pathway.; Doctor of Philosophy
- Publisher
- Federation University Australia
- Rights
- Copyright Alexander Nield
- Rights
- Open Access
- Rights
- This metadata is freely available under a CCO license
- Subject
- Zinc transporters; Insuling signalling
- Full Text
- Thesis Supervisor
- Myers, Mark
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